Device and methods for quantifying analytes

ABSTRACT

The present invention relates to devices and methods for measuring the quantity of multiple analytes in a sample. The device is designed such that each of the analyte sensing elements is configured to measure the quantity of a predetermined analyte and where the machine executable instructions are configured to select the proper analyte sensing element corresponding to the analyte to be measured.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/762,008, filed Jan. 25, 2006 and U.S. Provisional Application No.60/862,422, filed Oct. 20, 2006, the contents of which are incorporatedby reference as if set forth fully herein.

FIELD OF THE INVENTION

The present invention relates to devices and methods for measuring thequantity of an analyte in a sample using, for example, afluorescence-based assay, an absorbance-based assay, or alight-scattering assay.

BACKGROUND OF THE INVENTION

Conventional fluorometers used to perform analyte readings, aretypically designed as versatile instruments that can use several typesof excitation and emission filters and may be equipped with adjustablesensitivities, so that they may be configured for many different typesof assays. The Turner BioSystems TBS-380, and the BioRad VersaFluorfluorometer are examples of the typical laboratory fluorometer. Asignificant drawback to this design is that the user must choose thefilters and/or light sources to use, requiring the user to understandhow fluorescence works, look up the excitation and emission values oftheir assay, understand how to choose the appropriate filter sets andpossibly purchase and install new filter sets. In addition, the usermust often determine the appropriate gain setting (sensitivity) of theinstrument by an iterative process before beginning the assay. Theextensive tables that are offered with these instruments illustrate thepotential difficulties for the user in setting up the instrument toperform their assay of interest. In particular, if the user intends touse only one type of assay, this selection process presents a formidablebarrier to using the instrument.

In addition, conventional fluorometers typically measure light emittedfrom the sample and display the readout in relative fluorescence values.Because the display is in relative fluorescence values, the user must,in general, use standards to generate a standard curve, plot therelative fluorescence values of the standards, fit a line to the curve,compare the relative fluorescence value of the samples to the standardcurve, and ultimately back-calculate to determine the concentration ofthe sample. These operations can present difficulties to the untraineduser and, even for the experienced user, these operations are tediousand time-consuming. Generally, a fluorometer can be configured todownload data to a computer to make this operation easier.Unfortunately, this labor-saving feature requires installation ofsoftware onto a compatible computer, which may require purchasing acompatible computer, finding an appropriate communications port totransfer the data from the instrument to the computer, finding asuitable place in the laboratory where the instrument can permanently beconnected to the computer and then hoping that the installed softwarewill operate properly with the instrument. These actions can provideformidable barriers to the would-be user.

There is at least one fluorometer, the Turner BioSystems Modulusinstrument, which has some software built in for performing calculationsautomatically from standards provided by the user, making theperformance of those select assays easier for the user. The Turnerfluorometer, however, requires five standards to calculate the standardcurve, requiring a significant investment of time for the user, whichmay be particularly tedious if the user is measuring only a small numberof samples. Finally, this instrument is again designed for maximalflexibility, offering separate modules for each assay, which must besnapped into the instrument and are small enough to be easily lost in atypical laboratory environment.

Typical fluorometers also use specialized cuvettes to hold the sample.In general, the cuvettes are unique to a specific instrument, requireadapters for small sample sizes, are not generally available fromstandard laboratory supply companies and may be expensive.

What is desired in the art is a small device for the measurement of adefined set of assays. The device should be designed for seamlessintegration with the specific set of assays, such that theuser-interface would allow the user to choose from a defined set ofassays and immediately begin to perform the assay. Upon choosing theassay of interest, the device would automatically choose the correctlight sources, filter sets and sensitivity settings for the assaychosen. In addition, the device would be designed with sophisticatedalgorithms for data analysis appropriate for the specific assays, suchthat the customer need only measure a small number of standards (2 or3). The device would also be designed to calculate a standard curve fromthese standards, and upon measurement of the samples, the device wouldautomatically perform the required analysis and simply display theconcentration of the sample for the user. By building automaticconfigurations of light sources, filter sets and gain settings and byincorporating data analysis algorithms into the device, the user wouldno longer encounter a learning curve just to use the device. Inaddition, the user would not need to choose, purchase and installfilters, or determine the gain setting or sensitivity of the instrument.Finally, the user would be spared the tedium of using a large number ofstandards for the curve, plotting the curve, fitting a line to thecurve, comparing the value of the sample to the curve, andback-calculating the concentration of the sample from the standard curveequation. The device would have a small footprint and would not requireconnection to a computer, such that the instrument system would notrequire a large dedicated amount of benchspace. In addition, because thedevice would not require connection to a computer for data analysis, thedifficulties of finding a compatible computer for the software,installing the software on the computer and connecting the device to thecomputer are eliminated. Finally, the device would use areadily-available, inexpensive, disposable, laboratory test-tube tominimize the stress and expense of finding appropriate replacementcuvettes for the instrument.

SUMMARY OF THE INVENTION

The present invention relates to devices and methods for measuring thequantity of multiple analytes in a sample. In one embodiment, the devicecomprises a receptacle for holding a sample container having an analyteand optionally a reporter molecule, a photodetector, one or more analytesensing elements and a computer processing unit with machine executableinstructions. In turn, the analyte sensing elements comprise an energysource for exciting the sample, where the energy source is configured toemit a predetermined peak wavelength of light; an excitation filter thatisolates a predetermined range of wavelengths of light from the energysource; an emission filter that isolates a predetermined range ofwavelengths of light emitted from the excited sample. The device isdesigned such that each of the analyte sensing elements is configured tomeasure the quantity of a predetermined analyte and where the machineexecutable instructions are configured to select the proper analytesensing element corresponding to the analyte to be measured.

The present invention also relates to devices for measuring the quantityof an analyte in a sample, with the device comprising an energy source,a photodetector, a computer processing unit with machine executableinstructions and a receptacle for holding the sample tube, where thereceptacle is configured to fit a microcentrifuge tube.

The present invention also relates to methods of calculating thequantity of an analyte in a sample container, with the methodscomprising generating a fluorescence standard curve comprising measuringthe fluorescence intensity of a low-end or blank sample (g) andmeasuring the fluorescence intensity of at least one high-end standard(v), wherein the curve correlates fluorescence intensity to analytequantity, and wherein said curve has a predetermined degree ofsigmoidicity (n) and curvature (k). After generation of the fluorescencestandard curve, fluorescence intensity of said sample (y) is measured,where the sample comprises a fluorescent moiety capable of indicatingthe presence of the analyte in the sample. The fluorescence intensity ofthe sample (y) is then correlated to a quantity using said fluorescencestandard curve.

The present invention also relates to devices for measuring the ratio ofone analyte to another analyte in a sample, with the device comprisingspectrally dissimilar energy sources, one or more photodetectors able todistinguish the fluorescent emission from the two analytes, a computerprocessing unit with machine executable instructions and a receptaclefor holding the sample tube, where the receptacle is configured to fit amicrocentrifuge tube.

One aspect of the present invention provides a device for measuring thequantity of multiple analytes, said device comprising

-   -   a receptacle for holding a sample container having an analyte, a        photodetector, one or more analyte sensing elements and a        computer processing unit with machine executable instructions,        said analyte sensing element comprising:    -   a) an energy source for exciting said sample, wherein the energy        source is configured to emit a predetermined peak wavelength of        light;    -   b) an excitation filter, wherein said the excitation filter is        configured to isolate a predetermined range of wavelengths of        light from the energy source;    -   c) an emission filter, wherein the emission filter is configured        to isolate a predetermined range of wavelengths of light emitted        from the excited sample; and    -   wherein each of said analyte sensing elements is configured to        measure the quantity of a predetermined analyte and wherein said        machine executable instructions are configured to select the        proper analyte sensing element corresponding to the analyte to        be measured.

In a more particular embodiment, said energy source is a light emittingdiode. More particularly, said predetermined analyte is selected fromthe group consisting of DNA, RNA, protein, eukaryotic or prokaryoticcells, carbohydrates, lipids and metals ions. More particular still,said machine executable instructions are further configured to determinethe concentration of said specific analyte based upon an emitted lightfrom the excited sample. In another embodiment, said device furthercomprises a user interface. In another embodiment, said user interfacecomprises a display and a non-numerical keypad. In another embodiment,said sample container receptacle is configured to fit a 0.5microcentrifuge tube. In another embodiment, said device furthercomprises an internal power source. Another embodiment further comprisesat least one communications port. More particular still, saidcommunications port is selected from the group consisting of a universalserial bus (USB) port, an audio/video serial bus (IEEE 1394), aninfrared (IR) port and a radio frequency (RF) port.

In another embodiment, said device comprises a first and second analytesensing elements. More particularly, said first analyte sensing elementcomprises a diode that emits light with a peak wavelength of about 470nm, an excitation filter that filters out light with a wavelength ofgreater than about 490 nm and an emission filter that filters out lightwith a wavelength of less than about 520 nm and greater than about 580nm. More particular still, said second analyte sensing element comprisesa diode that emits light with a peak wavelength of about 640 nm, anexcitation filter that filters out light with a wavelength of less thanabout 570 and greater than about 647 nm and an emission filter thatfilters out light with a wavelength of less than about 652 nm.

In another embodiment, the user interface is configured to allow a userto select said analyte for measurement. More particularly, the machineexecutable instructions are capable of selecting said analyte formeasurement without user input.

In a more particular embodiment, the device is calibrated prior to thefirst use of said device by an end-user. Another embodiment furthercomprises a means for identifying an identity tag associated with saidsample container. More particularly, said identity tag ismachine-readable. More particular still, said identity tag is selectedfrom the group consisting of a barcode, a data matrix barcode and aradio frequency identification (RFID) tag.

Another embodiment of the invention provides a method of detecting ananalyte in a sample, said method comprising using the device describedherein.

Another aspect of the invention provides a method of calculating thequantity of an analyte in a sample container, said method comprising

-   -   a) generating a fluorescence standard curve comprising measuring        the fluorescence intensity of a blank sample (g) and measuring        the fluorescence intensity of at least one high-end standard        (v), wherein said curve correlates fluorescence intensity to        analyte quantity, and wherein said curve has a predetermined        degree of sigmoidicity (n) and curvature (k);    -   b) measuring the fluorescence intensity of said sample (y),        wherein said sample comprises a fluorescent moiety capable of        indicating the presence of said analyte in said sample; and    -   c) correlating said fluorescence intensity in said sample (y) to        the quantity of said analyte using said fluorescence standard        curve.

In another embodiment, said analyte quantity is the concentration ofsaid analyte. More particularly, said analyte is selected from the groupconsisting of DNA, RNA and protein. In another embodiment, saidfluorescence standard curve approaches linearity. In a more particularembodiment, (n) is approximately 1.

In another embodiment, the curve is characterized by the equation:

y=r(x ^(n)/(x ^(n) +k))+g;  (I)

wherein r is a correctional value determined by the formula:

r=(v−g)((s ^(n) +k)/s ^(n))  (II)

wherein (s) is the quantity of analyte in said high-end standard.

In another embodiment, said fluorescent moiety is a fluorescent compoundselected from the group consisting of cyanine and merocyanine dyes Moreparticularly, said the fluorescent moiety is selected from the groupconsisting of NanoOrange, OliGreen, PicoGreen, and RiboGreen.

In another embodiment, said high-end standard is present in said samplecontainer. More particularly, said high-end standard is immobilized ontoa solid surface.

In another embodiment, said solid surface is selected from the groupconsisting of the inner surface of said sample container, a bead, a chipand a fiber.

Another aspect of the invention provides a device for measuring thequantity of an analyte in a sample, said device comprising a computerprocessing unit with machine executable instructions that are configuredto perform the method described above.

In another more particular embodiment, said device further comprises:

-   -   a receptacle for holding a sample container having an analyte, a        photodetector and one or more analyte sensing elements, said        analyte sensing element comprising:    -   i) an energy source for exciting said sample, wherein the energy        source is configured to emit a predetermined peak wavelength of        light;    -   ii) an excitation filter, wherein said the excitation filter        isolates a predetermined range of wavelengths of light from the        energy source;    -   iii) an emission filter, wherein the emission filter isolates a        predetermined range of wavelengths of light emitted from the        excited sample; and    -   wherein each of said analyte sensing elements is configured to        measure the quantity of a predetermined analyte and wherein said        machine executable instructions are further configured to select        the proper analyte sensing element corresponding to the analyte        to be measured.

In another embodiment, said energy source is a light emitting diode. Inanother embodiment, said predetermined analyte is selected from thegroup consisting of DNA, RNA, cells, and protein.

In another said device further comprises a user interface. Moreparticularly, said user interface comprises a display and anon-numerical keypad. In another embodiment, said user interface isconfigured to allow a user to select said analyte for measurement. Inanother embodiment, said machine executable instructions are capable ofselecting said analyte for measurement without user input. In anotherembodiment, said device is calibrated prior to the first use of saiddevice by an end-user. In another embodiment, said sample containerreceptacle is configured to fit a 0.5 ml microcentrifuge tube.

In another embodiment, said device further comprises an internal powersource.

In another embodiment, said device comprises a first and second analytesensing elements. More particularly, said first analyte sensing elementcomprises a diode that emits light with a peak wavelength of about 470nm, an excitation filter that filters out light with a wavelength ofgreater than about 490 nm and an emission filter that filters out lightwith a wavelength of less than about 520 nm and greater than about 580nm. More particular still, said second analyte sensing element comprisesa diode that emits light with a peak wavelength of about 640 nm, anexcitation filter that filters out light with a wavelength of less thanabout 570 and greater than about 647 nm and an emission filter thatfilters out light with a wavelength of less than about 652 nm.

Another aspect of the invention provides a device for measuring thequantity of an analyte in a sample container, said device comprising anenergy source, a photodetector, a computer processing unit with machineexecutable instructions, and a receptacle for holding said samplecontainer, wherein said sample container comprises a polymer selectedfrom the group consisting of polypropylene and polyethylene.

In another embodiment, said energy source is a light emitting diode.

In another embodiment, said analyte is selected from the groupconsisting of DNA, RNA, protein, carbohydrates, lipids and metals ions.In another embodiment, said device further comprises a user interface.In another embodiment, said user interface comprises a display and anon-numerical keypad. More particularly, said user interface isconfigured to allow a user to select said analyte for measurement.

In another embodiment, said machine executable instructions are capableof selecting said analyte for measurement without user input. In anotherembodiment, said device further comprises an internal power source.

In another embodiment, the device further comprises at least onecommunications port. In another embodiment, said communications port isselected from the group consisting of a universal serial bus (USB) port,an audio/video serial bus (IEEE 1394), an infrared (IR) port and a radiofrequency (RF) port. In another embodiment, said device is calibratedprior to the first use of said device by an end-user.

In another embodiment, the device further comprises a means foridentifying an identity tag associated with said sample container. Inanother embodiment, said identity tag is machine-readable. In anotherembodiment, said identity tag is selected from the group consisting of abarcode, a data matrix barcode and a radio frequency identification(RFID) tag.

Another aspect of the invention provides a method of detecting ananalyte in a sample, said method comprising using the device describedabove.

Another aspect of the invention provides a method of calculating theratio of two analytes in a sample container, said method comprising

-   -   a) generating a fluorescence standard curve for the two analytes        comprising: measuring the fluorescence intensity of a first        blank analyte sample (g1) and a second analyte analyte sample        (g2) and measuring the fluorescence intensity of at least one        high-end standard for the first analyte (v1) and at least one        high-end standard for the second analyte (v2), wherein said        curve correlates fluorescence intensity to each analyte quantity        or relative quantity, and wherein said curves have a        predetermined degree of sigmoidicity (n) and curvature (k);    -   b) measuring the fluorescence intensity of said samples (y1 and        y2), wherein said sample comprises a fluorescent moiety capable        of indicating the presence of said analytes in said sample; and    -   c) correlating said fluorescence intensity in said samples (y1        and y2) to the quantity of said analyte using said fluorescence        standard curve.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a side view diagram of one embodiment of the presentinvention.

FIG. 2 depicts one embodiment of multiple analyte sensing units centeredon the sample container receptacle.

FIG. 3 depicts a theoretical fluorescence standard curve for RNA as theanalyte that can be generated using equation I. The high-end standardused to generate the curve contains 100 ng of RNA, the assay reliablygoes up to 200 ng when this 100 ng standard is used.

FIG. 4 depicts a device to measure the cleavage of a reporter from azone outside of the optical path of the instrument to one within theoptical path through diffusion or active mixing.

FIG. 5A depicts blue excitation signal as the ratio of Live:Dead cellsdecreases.

FIG. 5B depicts red excitation signal increasing as the ratio ofLive:Dead cells decrease.

FIG. 5C depicts ratio determination of Live:Dead cells.

FIG. 6 depicts Eukaryotic cell counting on the device described herein,wherein a fluorescent response is displayed for all eukaryotic celllines tested significantly above background.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The device and methods of present invention allow a seamless, intuitiveinteraction between the instrument and the user and accessibility of themethods to the user within the everyday workflow. Herein we disclose afluorometer that comprises an analyte sensing element (ASE) that isoperably linked allowing for detection of a predetermined analyte suchthat the selection of the predetermined analyte selects the appropriateASE. Thus, in one embodiment the device is configured such thatmachine-executable instructions select the proper analyte sensingelement that corresponds to the assay being used for detection of aspecific analyte. The methods comprise generating a standard curve bymeasuring two or more standards, one of which may be a zero or blankstandard. The standard curve can be generated by applying the values ofthe standard to a specific algorithm to generate an equation expressingthe relationship between the signal generated by the assay as read bythe instrument and the concentration of analyte in the sample. Thedevice may be designed such that the user interface prompts the user tochoose the assay, insert the standards, and insert the samples. Fromthis simple input, the device automatically can choose the appropriateanalyte-sensing elements and algorithm, perform the necessarycalculations to determine the standard curve, and compare the signalfrom the sample to the standard curve and perform the necessarycalculations to show the quantity of the analyte in the sample as areadout for the user. In addition, the device may also be configured toaccept an inexpensive, disposable plastic, optically clearmicrocentrifuge-shaped tube that holds the standards or samples and arereadily available. Furthermore, the device can be used to monitor andquantify multiple analytes in the same sample by, for example, labelinganalytes with different dyes and then exciting and/or filtering emissionspectra at particular wavelengths such that the dye/analyte of interestcan be distinctly monitored in the presence of other dyes/analytes.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a reporter molecule”includes a plurality of reporter molecules and reference to “afluorometer” includes a plurality of fluorometers and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

The term “analyte” refers to a molecule that is to be measured ordetected in the assay of this invention. The term “analyte” includes anysubstance for which there exists a specific binding molecule, or forwhich a specific binding molecule can be prepared, or for which theanalyte interacts with a reporter molecule to create a detectablesignal. Representative analytes include, but are not limited to, drugs,antigens, haptens, antibodies, proteins, peptides, amino acids,hormones, steroids, cancer cell markers, tissue cells, viruses,vitamins, nucleic acids, metal ions, enzymes, lipids, radioactiveisotopes, viruses, bacteria, pathogens, chemical contaminants, andpesticides.

The term “analyte sensing elements” or “ASE” as used herein refers to aparticular combination of 1) an energy source which itself may emit arestricted range of wavelengths of electromagnetic energy, 2) an“excitation filter” which is capable of isolating a range of wavelengthof electromagnetic energy, such as but not limited to light, and 3) an“emission filter” that is capable of isolating a range of wavelength ofelectromagnetic energy that is emitted from the sample wherein the threeparts are operably linked. The ASE are operably linked such that apredetermined analyte can be measured without the manual selection ofwavelength and filters or the need to perform additional calculations bythe end user.

The term “aqueous solution” as used herein refers to a solution that ispredominantly water and retains the solution characteristics of water.Where the aqueous solution contains solvents in addition to water, wateris typically the predominant solvent.

The term “buffer” as used herein refers to a system that acts tominimize the change in acidity or basicity of the solution againstaddition or depletion of chemical substances.

The term “detectable response” as used herein refers to an occurrenceof, or a change in, a signal that is directly or indirectly detectableeither by observation or by instrumentation. Typically, the detectableresponse is an optical response resulting in a change in the wavelengthdistribution patterns or intensity of absorbance or fluorescence or achange in light scatter, fluorescence lifetime, fluorescencepolarization, or a combination of these parameters. Alternatively, thedetectable response is an occurrence of a signal wherein the dye isinherently fluorescent and does not produce a change in signal uponbinding to a metal ion or phosphorylated target molecule. Alternatively,the detectable response is the result of a signal, such as color,fluorescence, radioactivity or another physical property of thedetectable label becoming spatially localized in a subset of a samplesuch as in a gel, on a blot, or an array, in a well of a microplate, ina microfluidic chamber, or on a microparticle as the result of formationof a ternary complex of the invention that comprises a phosphorylatedtarget molecule.

The term “energy source” as used herein refers to a light or wavelengthemitting device, preferably an LED, capable of exciting particles insolution.

The term “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound or metal ion, i.e., fluorogenic.Fluorophores may contain substitutents that alter the solubility,spectral properties or physical properties of the fluorophore. Numerousfluorophores are known to those skilled in the art and include, but arenot limited to coumarin, cyanine, benzofuran, a quinoline, aquinazolinone, an indole, a benzazole, a borapolyazaindacene andxanthenes including fluoroscein, rhodamine and rhodol as well assemiconductor nanocrystals and other fluorophores described in RICHARDP. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH CHEMICALS (10^(th) edition, 2005).

The term “kit” as used herein refers to a packaged set of relatedcomponents, typically one or more compounds or compositions.

The term “label” as used herein refers to a chemical moiety or proteinthat retains it's native properties (e.g. spectral properties,conformation and activity) when attached to a labeling reagent and usedin the present methods. The label can be directly detectable(fluorophore) or indirectly detectable (hapten or enzyme). Such labelsinclude, but are not limited to, radiolabels that can be measured withradiation-counting devices; pigments, dyes or other chromogens that canbe visually observed or measured with a spectrophotometer; spin labelsthat can be measured with a spin label analyzer; and fluorescent labels(fluorophores), where the output signal is generated by the excitationof a suitable molecular adduct and that can be visualized by excitationwith light that is absorbed by the dye or can be measured with standardfluorometers or imaging systems, for example. The label can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term label can also refer to a“tag” or hapten that can bind selectively to a conjugated molecule suchthat the conjugated molecule, when added subsequently along with asubstrate, is used to generate a detectable signal. For example, one canuse biotin as a tag and then use an avidin or streptavidin conjugate ofhorseradish peroxidate (HRP) to bind to the tag, and then use acolorimetric substrate (e.g., tetramethylbenzidine (TMB)) or afluorogenic substrate such as Amplex Red reagent (Molecular Probes,Inc.) to detect the presence of HRP. Numerous labels are know by thoseof skill in the art and include, but are not limited to, particles,fluorophores, haptens, enzymes and their colorimetric, fluorogenic andchemiluminescent substrates and other labels that are described inRICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH PRODUCTS (9^(th) edition, CD-ROM, September 2002), supra.

The term “machine executable instructions” as used herein refers to aset of instructions that cause a machine, such as a CPU, to perform amethod or assay.

The term “photodetector” as used herein refers to any device that iscapable of accepting an optical signal and producing an electricalsignal containing the same information as in the optical signal.

The term “predetermined analyte” as used herein refers to an analytethat is coordinated with an analyte sensing element (ASE) such thatselection of the predetermined analyte dictates the particular ASEpresent in the device.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 10 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues are an artificial chemical analogue of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers.

The term “sample” as used herein refers to any material that may containan analyte for detection or quantification. The analyte may include areactive group, e.g., a group through which a compound of the inventioncan be conjugated to the analyte. The sample may also include diluents,buffers, detergents, and contaminating species, debris and the like thatare found mixed with the target. Illustrative examples include urine,sera, blood plasma, total blood, saliva, tear fluid, cerebrospinalfluid, secretory fluids from nipples and the like. Also included aresolid, gel or semi-solid substances such as mucus, body tissues, cellsand the like suspended or dissolved in liquid materials such as buffers,extractants, solvents and the like. Typically, the sample is a livecell, a biological fluid that comprises endogenous host cell proteins,nucleic acid polymers, nucleotides, oligonucleotides, peptides,environmental material, food stuff, industrial material and buffersolutions. The sample may be in an aqueous solution, a viable cellculture or immobilized on a solid or semi solid surface such as a glassor plastic tube or cuvette.

Fluorometer and Methods of Use

One aspect of the present invention provides a device comprising areceptacle for holding a sample container having an analyte andoptionally a reporter molecule, a photodetector, one or more analytesensing elements and a computer processing unit with machine executableinstructions. In turn, the analyte sensing elements comprise an energysource for exciting the sample, where the energy source is configured toemit a predetermined peak wavelength of light; an excitation filter thatisolates a predetermined range of wavelengths of light from the energysource; an emission filter that isolates a predetermined range ofwavelengths of light emitted from the excited sample. The device isdesigned such that each of the analyte sensing elements is configured tomeasure the quantity of a predetermined analyte and where the machineexecutable instructions are configured to select the proper analytesensing element corresponding to the analyte to be measured.

FIG. 1 is a side view diagram of one embodiment of the presentinvention. FIG. 1 represents one embodiment of the architecture of theelements of the devices and it will become apparent to one of skill inthe art as to how to implement alternative architectures to achieve thefunctionality of the devices of the present invention. The systemillustrated in FIG. 1 comprises a receptacle for holding a samplecontainer 101, a power source 103, a computer processing unit 105, ananalyte sensing element which comprises an energy source 107, anexcitation filter 109 and an emission filter 111, and a photodetector113.

In one embodiment, the device is not limited by the sample container forwhich the receptacle is configured to receive. Example of samplecontainers that the receptacle may fit include but are not limited to,petri dishes, culture flasks, 4-well plates, 8-well plates, 24-wellplates, 96-well plates, cuvettes, centrifuge tubes, and microcentrifugetubes to name a few. As used herein a “receptacle being configured tofit or receive” means that the receptacle for the sample container isdesigned such that the sample container fits snuggly into the opening,allowing little or no movement of the sample container, beyond thevertical axis. In addition, the receptacle and the computer processingunit may or may not be coordinated to one another such that, unless thesample container is positioned properly in the receptacle, the computerprocessing unit will not initiate a measurement of the sample. Thereceptacle may accept only 1 sample container, or it may be configuredto accept 2, 3, 4, 5, 6, 7, 8, 9 10 or more sample tubes. In oneembodiment, the receptacle is configured to fit or receive amicrocentrifuge tube. Examples of microcentrifuge tubes are well know inthe art and include but are not limited to Eppendorf™ tubes,optically—clear microcentrifuge-shaped tubes such as those used inreal-time PCR experiments (an example is the Axygen PCR-05-C 500 μL PCRtube available from VWR) and generic centrifuge tubes. In a moreparticular embodiment, the receptacle may be configured to fit only onesize microcentrifuge tube or it may be configured to fit more than onesize of microcentrifuge tubes including, but not limited to, less than0.5 ml tubes, 0.5 ml tubes, 1.5 ml tubes, 2 ml tubes and greater than 2ml tubes.

Accordingly, in one embodiment, the present invention relates to adevice for measuring the quantity of an analyte in a sample container,where the device comprises an energy source, a photodetector, a computerprocessing unit with machine executable instructions and a receptaclefor holding the sample container, where the sample container comprisesan optically clear plastic. In one specific embodiment, the plastic iscomprised of polypropylene and/or polyethylene. Real-time PCRinstruments, such as the BioRad DNA Engine and the Opticon 2 Real-TimePCR Detection System are examples of instruments that use this type oftube for a sample requiring a fluorophore for detection. This type ofinstrument and the corresponding tubes are in common use, and thus thetubes are readily available from many sources.

The device of the present invention also comprises a photodetector. Thephotodetector can be any device that is capable of accepting an opticalsignal and producing an electrical signal containing the sameinformation as in the optical signal. Examples of photodetectors thatmay be used in the present invention include, but are not limited to,photoresistors, photovoltaic cells, photodiodes, photomultipliers,phototubes, phototransistors and pyroelectric devices that detectchanges in temperature due to illumination.

The device of the present invention also comprises one or more analytesensing elements. Each of the analyte sensing elements of the presentinvention comprise a particular combination of 1) an energy source whichitself may emit a restricted range of wavelengths of electromagneticenergy, 2) an “excitation filter” which is capable of isolating a rangeof wavelength of electromagnetic energy, such as but not limited tolight, and 3) an “emission filter” that is capable of isolating a rangeof wavelength of electromagnetic energy that is emitted from the sample.Upon excitation by energy from the energy source, the sample will,generally speaking, emit a form of electromagnetic energy, such as, butnot limited to light that can be generated by fluorescence,phosphorescence or luminescence. In one embodiment of the presentinvention, the device comprises a single analyte sensing element (ASE).In another embodiment, the device comprises more than ASE. In a moreparticular embodiment, the device comprises two, three, four, five, six,seven, eight, nine or ten or more ASEs. If the device comprises morethan one ASE, then the multiple ASEs may share one or more individualcomponents of the ASEs. Thus, for example, when a device of the presentinvention comprises two ASEs, these ASEs may share an energy source andhave separate emission filters and excitation filters. To continue theexample, the ASEs may share an energy source and an emission filter andhave separate excitation filters. Of course, in one embodiment, thedevice of the present invention may comprise more than one distinct ASE,where the distinct ASEs share neither an energy source nor an emissionfilter nor an excitation filter. In a more particular embodiment, thedevice comprises more than one ASE where none of components of the ASEsare shared, although they may be integrated into or connected to thesame computer processing unit.

As used herein, an energy source is a source of electromagnetic energyand includes any type of energy along the electromagnetic spectrumincluding, but not limited to, radio energy, microwave energy, infrared,visible light, ultraviolet light, x-ray light and even gamma radiation.In one embodiment, the energy emitted from the energy source is visiblelight. In a more particular embodiment, a peak wavelength of visiblelight is emitted from the energy source. For example, the peakwavelength of visible light may be, but is not limited to, between 400nm and 450 nm, or between 425 and 475 nm, or between 450 nm and 500 nm,or between 475 nm and 525 nm, or between 500 nm and 550 nm, or between525 and 575 nm, or between 550 nm and 600 nm, or between 575 nm and 625nm, or between 600 nm and 650 nm, or between 625 and 675 nm, or between650 nm and 700 nm, or between 675 nm and 725 nm. These peak wavelengthsideally correspond to the optimal excitation wavelength of the reportmolecule of choice for detection of a predetermined analyte. In anotherembodiment the peak wavelengths correspond to the optimal wavelength toproduct autofluorescence from the predetermined analyte. In yet anotherembodiment the peak wavelength correspond to the optimal wavelength formeasuring light scatter from the predetermined analyte.

The energy source can be any device or composition that is capable ofemitting electromagnetic energy. Examples of energy sources include, butare not limited to light emitting diodes (LED), incandescent lightbulbs, gas discharge lamps (e.g., helium, krypton, neon, argon, sodiumvapor and nitrogen), a laser, a maser, free charged particles such asions, accelerated particles, chemiluminescent chemicals, fluorescentsubstances, and phosphorescent substances. In one particular embodiment,the energy source is at least one light emitting diode. In anotherparticular embodiment, the energy source is more than one light emittingdiode. In a more particular embodiment, the energy source is a singlelight emitting diode that emits visible light. In an even moreparticular embodiment, the energy source is one or more light emittingdiodes that emits visible light with a predetermined peak wavelength.

Another component of the ASE of the present invention includes at leastone emission filter and one excitation filter. As used herein, anexcitation filter is a filter that is placed in between the energysource and the sample such that the energy emitted from the energysource is filtered prior to striking the sample. As used herein, theemission filter is a filter that is placed in between the sample and thephotodetector such that the energy emitted from the sample is filteredprior to striking the photodetector. In general, filters act to exclude(filter out) certain wavelengths of electromagnetic energy from passingthrough the filter. Filters may exclude wavelengths of electromagneticenergy below or above a specific wavelength. For example, a filter canexclude all wavelengths of light below 650 nm or all wavelengths above490 nm. Filters may also exclude electromagnetic energy within aspecific range of wavelengths. For example, a filter may exclude allwavelengths of light except for light with a wavelength in between about520 nm and about 580 nm. The selection of an appropriate filter for usewith the energy source should be readily apparent. In one specificembodiment, the ASE comprises an energy source that emits light, anexcitation filter that filters out light of wavelengths greater thanabout 490 nm and an emission filter than filters out light ofwavelengths less than about 520 nm and greater than about 580 nm. Inanother specific embodiment, the ASE comprises an energy source thatemits light, an excitation filter that filters out light of wavelengthsless than 570 nm and greater than about 647 nm and an emission filterthan filters out light of wavelengths of less than about 565 nm. In yetanother embodiment, the device comprises at least two ASEs where thefirst ASE comprises an energy source that emits light, an excitationfilter that filters out light of wavelengths greater than about 490 nmand an emission filter than filters out light of wavelengths less thanabout 520 nm and greater than about 580 nm, and where the second ASEcomprises an energy source that emits light, an excitation filter thatfilters out light of wavelengths less than 570 nm and greater than about647 nm and an emission filter than filters out light of wavelengths ofless than about 565 nm.

In one embodiment, the device comprises more than one ASE, and themultiple ASEs are configured in a spatial arrangement such that the corecomponents of the ASEs do not move. FIG. 2 depicts an example of adevice with 2 ASEs, where the core components of the ASEs are centeredon the sample receptacle. Referring to FIG. 2, the first ASE iscomprised of components 201, 203 and 207 and the second ASE is comprisedof components 213, 215 and 217. In one example of this spatialconfiguration, energy (e.g., light) is emitted from energy source 201(e.g., light emitting diode) and passes through excitation filter 203before passing through the sample that is sitting in receptacle 205.Once the light strikes the sample, the light emitted from the excitedsample passes through emission filter 207 and is reflected by mirror 209into or onto a photodetector 211. Continuing this example, energy (e.g.,light) is emitted from energy source 213 (e.g., light emitting diode)and passes through excitation filter 215 before passing through thesample that is sitting in receptacle 205. Once the light strikes thesample, the light emitted from the excited sample passes throughemission filter 217 and is reflected by mirror 219 into or onto aphotodetector 211. As is apparent from FIG. 2, mirrors may or may not benecessary to direct the energy beam into or onto the photodetector,depending on the spatial relationship between the energy emitted fromthe excited sample and the photodetector. Thus, one or more mirrors areoptional and may be present in some specific embodiments.

The devices of the present invention also comprise a computer processingunit. The processor controls the operation of the device and alsoprovides control of various functionalities of the device. The processorcan be a central processor that controls functionality via a busstructure or other communications interface. The processor can also beimplemented by distributing the processing functions among one or moreof the various components utilized to implement the functionalities ofthe devices.

One component of the computer processing unit will include memory.Memory is used to provide storage for program data or other data used bycomputer processing unit during operation and can be implemented usingvarious RAM or ROM memory devices. Memory can be used for example, tostore operating instructions and to provide memory registers foroperating and storage.

Memory can also be used in conjunction with a storage device such as,but not limited to, a disk storage device or a flash memory device. Astorage device can also be used to store program instructions, controland calibration curves, operational data, history logs, and other datawhich may be desired to be stored within the device. Alternatively, thestorage device, if one is present, need not be within the device. In oneembodiment, the storage device will not store large amounts of data, butthe data or instructions it stores is capable of being accessedfrequently and rapidly. In another embodiment, a cache is present tominimize latencies associated with retrieving frequently used data orinstructions from the storage device. In more specific embodiments, thestorage device may store less 1 gigabyte (GB), less than 500 megabytes(MB), less than 250 MB, less than 100 MB, less than 50 MB, less than 20MB, less than 10 MB, less than 9 MB, less than 8 MB, less than 7 MB,less than 6 MB, less than 5 MB, less than 4 MB, less than 3 MB, lessthan 2 MB or less than 1 MB of data and/or instructions. In anotherspecific embodiment, the storage device may store large amounts of data,for example 1 GB or more of data.

The memory of the device will comprise machine executable instructions.The machine executable instructions control the operation of the ASEswithin the device. For example, the machine executable instructions areconfigured to select an appropriate ASE, depending on the particularanalyte being measured. Thus, in one particular embodiment the devicecomprises more than one ASE and comprises machine executableinstructions. The end-user can select and input into the device thespecific analyte to be measured and, in turn, the machine executableinstructions will select and utilize the proper ASE within the device tomeasure the selected analyte. In a particular embodiment, the ASE hasbeen optimized for use with a specific reporter molecule, which is usedto measure the selected analyte.

For example, referring to FIG. 2, the end-user may select a specificanalyte to be measured by the device and the machine executableinstructions will determine which ASE to utilize. If one particularanalyte is chosen, the machine executable instructions will operate toturn on power to energy source 201 and photodetector 209, but not energysource 211 or photodetector 217. Energy from energy source 201 will passthrough excitation filter 203 and strike the sample sitting inreceptacle 205. Energy emitted from the sample will then pass throughemission filter 207 before traveling to photodetector 209. If theend-user then chooses a different analyte to measure, the machineexecutable instructions will operate to turn on power to energy source211 and photodetector 217, but not energy source 201 or photodetector209. Energy from energy source 211 will pass through excitation filter213 and strike the sample sitting in receptacle 205. Energy emitted fromthe sample will then pass through emission filter 215 before travelingto photodetector in 217. In this sense, the machine executableinstructions are capable of being configured to select the proper ASEthat corresponds to the analyte being measured.

In another embodiment, the machine executable instructions areconfigured such that they are capable of selecting the analyte to bemeasured, without end-user input. In this embodiment, the end-used willsimply place the sample container in receptacle 205. Once in place, themachine executable instructions may or may not perform one or a seriesof operations to determine the most appropriate ASE to use to analyzethe sample. Once the machine executable instructions select the properASE that corresponds to the analyte within the sample, the machineexecutable instructions then performs the assay to determine analyteconcentration.

In yet another embodiment, the machine executable instructions may alsocomprise calibration data, such as but not limited to, calibration curvedata, internal standard data and the like. For example, the calibrationdata may be written into the machine executable instructions such thatthere is not a need for the end-user to acquire blank and standardmeasurement data. The machine executable instructions may thus allow thedevice to be calibrated prior to the first use of an end-user. And themachine executable instructions may also allow the device to be entirely“calibration free” in relation to the end-user.

In another embodiment the device monitors and quantifies multipleanalytes in the same sample by, for example, labeling analytes withdifferent dyes and then exciting and/or filtering emission spectra atparticular wavelengths such that the dye/analyte of interest can bedistinctly monitored in the presence of other dyes/analytes.Accordingly, a particular embodiment of the present invention providesfor simultaneous monitoring of multiple analytes, such as by concomitantdetection of multiple dyes/analytes in a single sample.

The devices of the present invention are designed to measure thequantity of multiple analytes in a sample. The devices may be designedto measure the multiple analytes simultaneously, or the devices may beconfigured to measure the analytes “one at a time.” The analytes to bequantified may be any analyte, provided the device is configured tomeasure the specific analyte desired. As used herein, an analyte is achemical, composition or an organism in a sample that is to be analyzed.Examples of analytes to be quantified include, but are not limited tonucleic acids such as DNA and RNA, proteins, carbohydrates, lipids,proteoglycans, glycoproteins, proteolipids, lipoproteins, metal ions,prokaryotic and eukaryotic cells, and viral particles. In one specificembodiment, the device is capable of quantifying DNA, RNA, eukaryoticand prokaryotic cells, and protein.

In one embodiment the selected analyte is measured using a reportermolecule. The term “reporter molecule” as used herein refers to anyluminescent molecule that is capable of producing a visible signal whenassociated with an anlayte, either directly or indirectly. Included arereporter typically used in a fluorometer for detection of an analytesuch as nucleic acid and proteins. Reporter molecules that are presentlycommercially available include, but are not limited to, the dyes inQuant-It® kits (Invitrogen), Sypro® dyes, Picogreen® dye, Deep Purpleprotein stain, Syto® Dyes, Sybr® dyes, Flamingo® dyes, and Lucy® dyes.Typically, luminescent molecules, as used herein include dyes,fluorescent proteins, phosphorescent dyes, chromophores, enzymesubstrates, haptens and chemiluminescent compounds particles, haptens,enzymes and their colorimetric, fluorogenic and chemiluminescentsubstrates that are capable of producing a detectable signal uponappropriate activation. The term “dye” refers to a compound that emitslight to produce an observable detectable signal. “Dye” includesfluorescent and non-fluorescent compounds that include withoutlimitations pigments, fluorophores, chemiluminescent compounds,luminescent compounds and chromophores. The term “chromophore” as usedherein refers to a label that emits and/or reflects light in the visiblespectra that can be observed without the aid of instrumentation. Theterm “fluorophore” as used herein refers to a composition that isinherently fluorescent or demonstrates a change in fluorescence uponbinding to a biological compound, i.e. can be fluorogenic or theintensity can be diminished by quenching. Fluorophores may containsubstitutents that alter the solubility, spectral properties or physicalproperties of the fluorophore. Numerous fluorophores are known to thoseskilled in the art and include, but are not limited to coumarin,cyanine, benzofuran, a quinoline, a quinazolinone, an indole, abenzazole, a borapolyazaindacene and xanthenes including fluoroscein,rhodamine and rhodol as well as other fluorophores described in RICHARDP. HAUGLAND, The Handbook, A Guide to Fluorescent Probes and LabelingTechnologies (10^(th) edition, 2005).

Numerous fluorogenic and colorimetric enzyme substrates exist for theamplification of a signal as well as substrates used to directly detectthe function of an anlyte, e.g. enzymes that cleave the substrateresulting in a detectable signal. Both are included in the presentinvention for the detection of a predetermined analyte. In the case ofthe enzyme substrate used to amplify the signal the analyte isassociated with an enzyme. In the case where the enzyme substratedirectly detects the ananlyte, the analyte is the enzyme. Colorimetricor fluorogenic substrate and enzyme combination included, but are notlimited to, uses of oxidoreductases such as horseradish peroxidase and asubstrate such as 3,3′-diaminobenzidine (DAB) and3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color(brown and red, respectively). Other colorimetric oxidoreductasesubstrates that yield detectable products include, but are not limitedto: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPD), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 4-chloro-1-naphthol. Fluorogenicsubstrates include, but are not limited to, homovanillic acid or4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reducedbenzothiazines, including Amplex® Red and Amplex Ultra Red reagent andits variants (U.S. Pat. No. 4,384,042 and U.S. Ser. No. 10/980,139) andreduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat. No.6,162,931) and dihydrorhodamines including dihydrorhodamine 123.Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306;5,583,001 and 5,731,158) represent a unique class of peroxidasesubstrates in that they can be intrinsically detectable before action ofthe enzyme but are “fixed in place” by the action of a peroxidase in theprocess described as tyramide signal amplification (TSA). Thesesubstrates are extensively utilized to label targets in samples that arecells, tissues or arrays for their subsequent detection by microscopy,flow cytometry, optical scanning and fluorometry.

Another preferred colorimetric (and in some cases fluorogenic) substrateand enzyme combination uses a phosphatase enzyme such as an acidphosphatase, an alkaline phosphatase or a recombinant version of such aphosphatase in combination with a colorimetric substrate such as5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolylphosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenylphosphate, or o-nitrophenyl phosphate or with a fluorogenic substratesuch as 4-methylumbelliferyl phosphate,6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat.No. 5,830,912) fluorescein diphosphate, 3-O-methylfluorescein phosphate,resorufin phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates(U.S. Pat. Nos. 5,316,906 and 5,443,986).

Glycosidases, in particular beta-galactosidase, beta-glucuronidase andbeta-glucosidase, are additional suitable enzymes. Appropriatecolorimetric substrates include, but are not limited to,5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similarindolyl galactosides, glucosides, and glucuronides, o-nitrophenylbeta-D-galactopyranoside (ONPG) and p-nitrophenylbeta-D-galactopyranoside. Preferred fluorogenic substrates includeresorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG),fluorescein diglucuronide and their structural variants (U.S. Pat. Nos.5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236),4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferylbeta-D-galactopyranoside and fluorinated coumarinbeta-D-galactopyranosides (U.S. Pat. No. 5,830,912).

In another embodiment, enzyme substrates used to detect the presence ofenzymes associated with microbiral resistance to antibiotics, such asβ-lactam, include beta-lactamase substrates, including, but not limitedto, any substrates and method of use disclosed in U.S. Ser. No.11/040,924; and US20030003526.

Additional enzymes include, but are not limited to, hydrolases such ascholinesterases and peptidases, oxidases such as glucose oxidase andcytochrome oxidases, and reductases for which suitable substrates areknown.

Enzymes and their appropriate substrates that produce chemiluminescenceare preferred for some assays. These include, but are not limited to,natural and recombinant forms of luciferases and aequorins.Chemiluminescence-producing substrates for phosphatases, glycosidasesand oxidases such as those containing stable dioxetanes, luminol,isoluminol and acridinium esters are additionally useful.

In addition to enzymes, haptens such as biotin are also preferredlabels. Biotin is useful because it can function in an enzyme system tofurther amplify the detectable signal, and it can function as a tag tobe used in affinity chromatography for isolation purposes. For detectionpurposes, an enzyme conjugate that has affinity for biotin is used, suchas avidin-HRP. Subsequently a peroxidase substrate is added to produce adetectable signal.

Haptens also include hormones, naturally occurring and synthetic drugs,pollutants, allergens, affector molecules, growth factors, chemokines,cytokines, lymphokines, amino acids, peptides, chemical intermediates,nucleotides and the like.

In a separate embodiment the reporter molecule is a dye or label that isconjugated to a specific binding partner, wherein the specific bindingpartner binds to the analyte or a molecule covalently attached to theanalyte. The term “label” as used herein refers to a chemical moiety orprotein that retains it's native properties (e.g. spectral properties,conformation and activity) when attached to a labeling reagent and usedin the present methods. The label can be directly detectable(fluorophore) or indirectly detectable (hapten or enzyme). Such labelsinclude, but are not limited to, radiolabels that can be measured withradiation-counting devices; pigments, dyes or other chromogens that canbe visually observed or measured with a spectrophotometer; spin labelsthat can be measured with a spin label analyzer; and fluorescent labels(fluorophores), where the output signal is generated by the excitationof a suitable molecular adduct and that can be visualized by excitationwith light that is absorbed by the dye or can be measured with standardfluorometers or imaging systems, for example. The label can be achemiluminescent substance, where the output signal is generated bychemical modification of the signal compound; a metal-containingsubstance; or an enzyme, where there occurs an enzyme-dependentsecondary generation of signal, such as the formation of a coloredproduct from a colorless substrate. The term label can also refer to a“tag” or hapten that can bind selectively to a conjugated molecule suchthat the conjugated molecule, when added subsequently along with asubstrate, is used to generate a detectable signal. For example, one canuse biotin as a tag and then use an avidin or streptavidin conjugate ofhorseradish peroxidate (HRP) to bind to the tag, and then use acolorimetric substrate (e.g., tetramethylbenzidine (TMB)) or afluorogenic substrate such as Amplex Red reagent (Molecular Probes,Inc.) to detect the presence of HRP. Numerous labels are know by thoseof skill in the art and include, but are not limited to, particles,fluorophores, haptens, enzymes and their colorimetric, fluorogenic andchemiluminescent substrates and other labels that are described inRICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH PRODUCTS (9^(th) edition, CD-ROM, September 2002), supra.

Typically the label would be an antibody, antigen, biotin orstreptavidin, all conjugates typically used in an immunoassay. However,there is no intended limitation of the specific binding partner that canbe conjugated to a label and used in the present methods to detect atarget analyte.

TABLE 2 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor folate folate binding protein toxin toxin receptorcarbohydrate lectin or carbohydrate receptor peptide peptide receptorprotein protein receptor enzyme substrate enzyme Fc region Anti-Fcantibody hormone hormone receptor ion chelator

In a particular aspect the carrier molecule is an antibody fragment,such as, but not limited to, anti-Fc, an anti-Fc isotype, anti-J chain,anti-kappa light chain, anti-lambda light chain, or a single-chainfragment variable protein; or a non-antibody peptide or protein, suchas, for example but not limited to, soluble Fc receptor, protein G,protein A, protein L, lectins, or a fragment thereof. In one aspect thecarrier molecule is a Fab fragment specific to the Fc portion of thetarget-binding antibody or to an isotype of the Fc portion of thetarget-binding antibody (U.S. Ser. No. 10/118,204). The monovalent Fabfragments are typically produced from either murine monoclonalantibodies or polyclonal antibodies generated in a variety of animals,for example but not limited to, rabbit or goat. These fragments can begenerated from any isotype such as murine IgM, IgG₁, IgG_(2a), IgG_(2b)or IgG₃.

Alternatively, a non-antibody protein or peptide such as protein G, orother suitable proteins, can be used alone or coupled with albumin.Preferred albumins include human and bovine serum albumins or ovalbumin.Protein A, G and L are defined to include those proteins known to oneskilled in the art or derivatives thereof that comprise at least onebinding domain for IgG, i.e. proteins that have affinity for IgG. Theseproteins can be modified but do not need to be and are conjugated to areactive label in the same manner as the other carrier molecules of theinvention.

In another aspect the carrier molecule is a whole intact antibody.Antibody is a term of the art denoting the soluble substance or moleculesecreted or produced by an animal in response to an antigen, and whichhas the particular property of combining specifically with the antigenthat induced its formation. Antibodies themselves also serve areantigens or immunogens because they are glycoproteins and therefore areused to generate anti-species antibodies. Antibodies, also known asimmunoglobulins, are classified into five distinct classes—IgG, IgA,IgM, IgD, and IgE. The basic IgG immunoglobulin structure consists oftwo identical light polypeptide chains and two identical heavypolypeptide chains (linked together by disulfide bonds).

When IgG is treated with the enzyme papain a monovalent antigen-bindingfragment can be isolated, referred herein to as a Fab fragment. When IgGis treated with pepsin (another proteolytic enzyme), a larger fragmentis produced, F(ab′)₂. This fragment can be split in half by treatingwith a mild reducing buffer that results in the monovalent Fab′fragment. The Fab′ fragment is slightly larger than the Fab and containsone or more free sulfhydryls from the hinge region (which are not foundin the smaller Fab fragment). The term “antibody fragment” is usedherein to define the Fab′, F(ab′)₂ and Fab portions of the antibody. Itis well known in the art to treat antibody molecules with pepsin andpapain in order to produce antibody fragments (Gorevic et al., Methodsof Enzyol., 116:3 (1985)).

The monovalent Fab fragments of the present invention are produced fromeither murine monoclonal antibodies or polyclonal antibodies generatedin a variety of animals that have been immunized with a foreign antibodyor fragment thereof, U.S. Pat. No. 4,196,265 discloses a method ofproducing monoclonal antibodies. Typically, secondary antibodies arederived from a polyclonal antibody that has been produced in a rabbit orgoat but any animal known to one skilled in the art to producepolyclonal antibodies can be used to generate anti-species antibodies.The term “primary antibody” describes an antibody that binds directly tothe antigen as opposed to a “secondary antibody” that binds to a regionof the primary antibody. Monoclonal antibodies are equal, and in somecases, preferred over polyclonal antibodies provided that theligand-binding antibody is compatible with the monoclonal antibodiesthat are typically produced from murine hybridoma cell lines usingmethods well known to one skilled in the art.

In one aspect the antibodies are generated against only the Fc region ofa foreign antibody. Essentially, the animal is immunized with only theFc region fragment of a foreign antibody, such as murine. The polyclonalantibodies are collected from subsequent bleeds, digested with anenzyme, pepsin or papain, to produce monovalent fragments. The fragmentsare then affinity purified on a column comprising whole immunoglobulinprotein that the animal was immunized against or just the Fc fragments.

The labels of the present invention include any directly or indirectlydetectable label known by one skilled in the art that can be covalentlyattached to a specific binding partner. Labels include, withoutlimitation, a chromophore, a fluorophore, a fluorescent protein, aphosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and aradioisotope. Preferred labels include fluorophores, fluorescentproteins, haptens, and enzymes.

A fluorophore of the present invention is any chemical moiety thatexhibits an absorption maximum beyond 280 nm, and when covalentlyattached to a labeling reagent retains its spectral properties.Fluorophores of the present invention include, without limitation; apyrene (including any of the corresponding derivative compoundsdisclosed in U.S. Pat. No. 5,132,432), an anthracene, a naphthalene, anacridine, a stilbene, an indole or benzindole, an oxazole orbenzoxazole, a thiazole or benzothiazole, a4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a cyanine (including anycorresponding compounds in U.S. Ser. Nos. 09/968,401 and 09/969,853), acarbocyanine (including any corresponding compounds in U.S. Ser. Nos.09/557,275; 09/969,853 and 09/968,401; U.S. Pat. Nos. 4,981,977;5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044;5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134;6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO99/51702, WO 01/21624; EP 1 065 250 A1), a carbostyryl, a porphyrin, asalicylate, an anthranilate, an azulene, a perylene, a pyridine, aquinoline, a borapolyazaindacene (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113;and 5,433,896), a xanthene (including any corresponding compoundsdisclosed in U.S. Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392;5,451,343 and U.S. Ser. No. 09/922,333), an oxazine (including anycorresponding compounds disclosed in U.S. Pat. No. 4,714,763) or abenzoxazine, a carbazine (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,810,636), a phenalenone, a coumarin(including an corresponding compounds disclosed in U.S. Pat. Nos.5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran (includingan corresponding compounds disclosed in U.S. Pat. Nos. 4,603,209 and4,849,362) and benzphenalenone (including any corresponding compoundsdisclosed in U.S. Pat. No. 4,812,409) and derivatives thereof. As usedherein, oxazines include resorufins (including any correspondingcompounds disclosed in 5,242,805), aminooxazinones, diaminooxazines, andtheir benzo-substituted analogs.

When the fluorophore is a xanthene, the fluorophore is optionally afluorescein, a rhodol (including any corresponding compounds disclosedin U.S. Pat. Nos. 5,227,487 and 5,442,045), or a rhodamine (includingany corresponding compounds in U.S. Pat. Nos. 5,798,276; 5,846,737; U.S.Ser. No. 09/129,015). As used herein, fluorescein includes benzo- ordibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins.Similarly, as used herein rhodol includes seminaphthorhodafluors(including any corresponding compounds disclosed in U.S. Pat. No.4,945,171). Alternatively, the fluorophore is a xanthene that is boundvia a linkage that is a single covalent bond at the 9-position of thexanthene. Preferred xanthenes include derivatives of3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of6-amino-3H-xanthen-3-imine attached at the 9-position.

Preferred fluorophores of the invention include xanthene (rhodol,rhodamine, fluorescein and derivatives thereof) coumarin, cyanine,pyrene, oxazine and borapolyazaindacene. Most preferred are sulfonatedxanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinatedcoumarins and sulfonated cyanines. The choice of the fluorophoreattached to the specific binding partner will determine the absorptionand fluorescence emission properties of the reporter molecule andsubsequent selection of the ASE. Physical properties of a fluorophorelabel include spectral characteristics (absorption, emission and stokesshift), fluorescence intensity, lifetime, polarization andphoto-bleaching rate all of which can be used to distinguish onefluorophore from another.

Typically the fluorophore contains one or more aromatic orheteroaromatic rings, that are optionally substituted one or more timesby a variety of substituents, including without limitation, halogen,nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl,cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, orother substituents typically present on fluorophores known in the art.

In one aspect of the invention, the fluorophore has an absorptionmaximum beyond 480 nm. In a particularly useful embodiment, thefluorophore absorbs at or near 488 nm to 514 nm (particularly suitablefor excitation by the output of the argon-ion laser excitation source)or near 546 nm (particularly suitable for excitation by a mercury arclamp).

Many of fluorophores can also function as chromophores and thus thedescribed fluorophores are also preferred chromophores of the presentinvention.

Fluorescent proteins may also find use as labels in the presentinvention. Examples of fluorescent proteins include green fluorescentprotein (GFP) and the phycobiliproteins and the derivatives thereof. Thefluorescent proteins, especially phycobiliprotein, are particularlyuseful for creating tandem dye labeled labeling reagents. These tandemdyes comprise a fluorescent protein and a fluorophore for the purposesof obtaining a larger stokes shift wherein the emission spectra isfarther shifted from the wavelength of the fluorescent protein'sabsorption spectra. This is particularly advantageous for detecting alow quantity of a target in a sample wherein the emitted fluorescentlight is maximally optimized, in other words little to none of theemitted light is reabsorbed by the fluorescent protein. For this towork, the fluorescent protein and fluorophore function as an energytransfer pair wherein the fluorescent protein emits at the wavelengththat the fluorophore absorbs at and the fluorphore then emits at awavelength farther from the fluorescent proteins than could have beenobtained with only the fluorescent protein. A particularly usefulcombination is the phycobiliproteins disclosed in U.S. Pat. Nos.4,520,110; 4,859,582; 5,055,556, and the sulforhodamine fluorophoresdisclosed in 5,798,276, or the sulfonated cyanine fluorophores disclosedin U.S. Ser. Nos. 09/968/401 and 09/969/853; or the sulfonated xanthenederivatives disclosed in 6,130,101, and those combinations disclosed inU.S. Pat. No. 4,542,104. Alternatively, the fluorophore functions as theenergy donor and the fluorescent protein is the energy acceptor.

In one embodiment, the label is a fluorophore selected from the groupconsisting of fluorescein, coumarins, rhodamines, 5-TMRIA(tetramethylrhodamine-5-iodoacetamide),(9-(2(or4)-(N-(2-maleimdylethyl)-sulfonamidyl)-4(or2)-sulfophenyl)-2,3,6,7,12,13,16,17-octahydro-(1H,5H,11H,15H-xantheno(2,3,4-ij:5,6,7-i′j′)diquinolizin-18-iumsalt) (Texas Red®),2-(5-(1-(6-(N-(2-maleimdylethyl)-amino)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1,3-propyldienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indoliumsalt (Cy™3),N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine(IANBD amide), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan),pyrene,6-amino-2,3-dihydro-2-(2-((iodoacetyl)amino)ethyl)-1,3-dioxo-1H-benz(de)isoquinoline-5,8-disulfonicacid salt (lucifer yellow),2-(5-(1-(6-(N-(2-maleimdylethylyamino)-6-oxohexyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1,3-pentadienyl)-1-ethyl-3,3-dimethyl-5-sulfo-3H-indoliumsalt (Cy™5),4-(5-(4-dimethylaminophenyl)oxazol-2-yl)phenyl-N-(2-bromoacetamidoethyl)sulfonamide(Dapoxyl® (2-bromoacetamidoethyl)sulfonamide)),(N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide(BODIPY® 507/545 IA),N-(4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N′-iodoacetylethylenediamine(BODIPY 530/550 IA),5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid(1,5-IAEDANS), and carboxy-X-rhodamine, 5/6-iodoacetamide (XRIA 5,6).Another example of a label is BODIPY-FL-hydrazide. Other luminescentlabels include lanthanides such as europium (Eu3+) and terbium (Tb3+),as well as metal-ligand complexes of ruthenium [Ru(II)], rhenium[Re(I)], or osmium [0s(II)], typically in complexes with diimine ligandssuch as phenanthroline.

In another embodiment the reporter molecules are fluorgenic wherein theybecome fluorescent when associated with the analyte. Such reportermolecules include dyes that associate with nucleic acid (DNA and/orRNA), proteins (total and subsets such as post-translationaly modifiedproteins), pH, and metal ions. Reporter molecules for the detection ofnucleic acid typically include unsymmetrical cyanine compounds, eithermonomers or dimmers, including, but not limited to compounds disclosedin U.S. Pat. Nos. 4,957,870; 4,883,867; 5,436,134; 5,658,751, 5,534,416;5,863,753; 5,410,030; 5,582,977; 6,664,047; U.S. Ser. Nos. 10/911,423;11/005,860; 11/005,861; 60/680,243 and WO 93106482; ethidium dimers(U.S. Pat. No. 5,314,805), acridine dimers and acridine-ethidiumheterodimers (U.S. Pat. No. 6,428,667 and Rye, et al. Nucleic AcidsResearch (1990) 19(2), 327). The following references describe DNAintercalating fluorescent dimers and their physical characteristics:Gaugain et al., Biochemistry (1978) 17:5071-5078; Gaugain et al.,Biochemistry (1978) 17:5078-5088; Markovits et al., Anal. Biochemistry(1979) 94:259-269; Markovits et al. Biochemistry (1983) 22: 3231-3237;and Markovits et al., Nucl. Acids Res. (1985) 13:3773-3788. Commerciallyavailable dyes include PicoGreen, RiboGreen and OliGreen (Invitrogen).

In another embodiment the reporter molecules stain proteins, eitherdirectly or by forming a ternary complex comprising a metal ion. Suchreporter molecules include, but are not limited to those disclosed inU.S. Pat. No. 5,616,502; U.S. Ser. Nos. 11/241,323; 11/199,641;11/063,707; 10/966,536; 10/703,816; and 6,967,251. Commerciallyavailable dyes include NanoOrange, and Coomassie Fluor (Invitrogen).

In another embodiment the reporter molecules become fluorescent afterassociating with ions. Such reporter molecules include, but are notlimited to those disclosed in U.S. Pat. Nos. 6,316,267; 6,162,931;5,648,270; 6,013,802; 5,405,975; 5,516,864; 5,453,517; 6,962,992; U.S.Ser. Nos. 10/634,336; and 11/191,799. Commercially available dyesinclude fluo-3, fluo-4, Corona Red, Corona Green, Leadmuin Green, FuraCalcium Indicators.

Particular Aspects of the Invention:

One aspect of the present invention provides device for measuring thequantity of one or more predetermined analytes using a reportermolecule, wherein the device is an integrated unit which comprises

-   -   a receptacle for holding a sample container having an analyte        and the reporter molecule, a photodetector, one or more fixed        and distinct analyte sensing elements (ASE) and a computer        processing unit with machine executable instructions, wherein        the ASE comprises:    -   a) an energy source for exciting the sample, wherein the energy        source is configured to emit a predetermined peak wavelength of        light;    -   b) an excitation filter, wherein the excitation filter is        configured to isolate a predetermined range of wavelengths of        light from the energy source;    -   c) an emission filter, wherein the emission filter is configured        to isolate a predetermined range of wavelengths of light emitted        from the excited sample; and    -   wherein each of the ASE is configured to measure the quantity of        the predetermined analyte and wherein the machine executable        instructions are configured to select the proper ASE        corresponding to the analyte to be measured.

In a preferred embodiment, the energy source is a light emitting diode(LED).

In another embodiment, the predetermined analyte is selected from thegroup consisting of DNA, RNA, protein, eukaryotic or prokaryotic cells,carbohydrates, lipids, viruses, pH and metals ions. In anotherembodiment, the machine executable instructions are further configuredto determine the concentration of the specific analyte based upon anemitted light from the excited sample. In another embodiment, the devicefurther comprises a user interface. In another embodiment, the userinterface comprises a display and a non-numerical keypad.

In another embodiment, the sample container receptacle is configured tofit an optically clear 0.5 microcentrifuge tube. In another embodiment,the device further comprises an internal power source. In anotherembodiment, the device further comprises at least one communicationsport. More particularly, the communications port is selected from thegroup consisting of a universal serial bus (USB) port, an audio/videoserial bus (IEEE 1394), an infrared (IR) port and a radio frequency (RF)port.

In another embodiment, the device comprises a first and second ASE. Moreparticularly, the first ASE comprises a LED that emits light with a peakwavelength of about 470 nm, an excitation filter that filters out lightwith a wavelength of greater than about 490 nm and an emission filterthat filters out light with a wavelength of less than about 520 nm andgreater than about 580 nm. More particular still, the second ASEcomprises a LED that emits light with a peak wavelength of about 640 nm,an excitation filter that filters out light with a wavelength of lessthan about 570 and greater than about 647 nm and an emission filter thatfilters out light with a wavelength of less than about 652 nm.

In another embodiment, the user interface is configured to allow a userto select the analyte for measurement.

In another embodiment, the machine executable instructions are capableof selecting the analyte for measurement without user input.

The device of claim 1, wherein dimensions of the device are about 30-300mm on a minor axis, 100-500 mm on a major axis, and a variable thicknessof about 10 to 100 mm; with the proviso that the dimension of the majoraxis is greater than the minor axis.

Another aspect of the invention provides a method of calculating thequantity of an analyte in an optically clear sample container, themethod comprising

-   -   a) generating a fluorescence standard curve comprising measuring        the fluorescence intensity of a blank sample (g) and measuring        the fluorescence intensity of at least one high-end standard        (v), wherein the curve correlates fluorescence intensity to        analyte quantity, and wherein the curve has a predetermined        degree of sigmoidicity (n) and curvature (k);    -   b) measuring the fluorescence intensity of the sample (y),        wherein the sample comprises a fluorescent moiety capable of        indicating the presence of the analyte in the sample; and    -   c) correlating the fluorescence intensity in the sample (y) to        the quantity of the analyte using the fluorescence standard        curve.

In another embodiment, the curve is characterized by the equation:

y=r(x ^(n)/(x ^(n) +k))+g;  (I)

wherein r is a correctional value determined by the formula:

r=(v−g)((s ^(n) +k)/s ^(n))  (II)

wherein (s) is the quantity of analyte in the high-end standard.

Another embodiment provides a method for detecting the presence of apredetermined analyte in a sample wherein the sample is in an opticallyclear sample container, the method comprising:

-   -   a) contacting the sample with a reporter molecule to form a        contacted sample:    -   b) detecting the presence of the predetermine analyte, wherein        detecting comprises placing the optically clear container in a        receptacle for holding the sample container of an integrated        device for the measurement of a predetermined analyte, wherein        the device further comprises:        -   i) a photodetector, one or more fixed and distinct analyte            sensing elements (ASE) and a computer processing unit with            machine executable instructions, wherein the ASE comprises:            -   an energy source for exciting the sample, wherein the                energy source is configured to emit a predetermined peak                wavelength of light;            -   an excitation filter, wherein the excitation filter is                configured to isolate a predetermined range of                wavelengths of light from the energy source;            -   an emission filter, wherein the emission filter is                configured to isolate a predetermined range of                wavelengths of light emitted from the excited sample;                and        -   wherein each of the ASE is configured to measure the            quantity of the predetermined analyte and wherein the            machine executable instructions are configured to select the            proper ASE corresponding to the analyte to be measured.

Another aspect of the invention provides a method of calculating theratio of two analytes in a sample container, the method comprising

-   -   a) generating a fluorescence standard curve for the two analytes        comprising: measuring the fluorescence intensity of a first        blank analyte sample (g1) and a second analyte sample (g2) and        measuring the fluorescence intensity of at least one high-end        standard for the first analyte (v1) and at least one high-end        standard for the second analyte (v2), wherein the curve        correlates fluorescence intensity to each analyte quantity or        relative quantity, and wherein the curves have a predetermined        degree of sigmoidicity (n) and curvature (k);    -   b) measuring the fluorescence intensity of the samples (y1 and        y2), wherein the sample comprises a fluorescent moiety capable        of indicating the presence of the analytes in the sample; and    -   c) correlating the fluorescence intensity in the samples (y1 and        y2) to the quantity of the analyte using the fluorescence        standard curve.

Another embodiment of the invention provides, a method of detecting anenzyme or cleavage substrate in a sample, the method comprising:

-   -   providing a device as described herein comprising a receptacle        comprising a label bound to a cleavable linker, wherein the        cleavable linker is cleaved by the enzyme or cleavage substrate;    -   adding a sample suspected of containing the enzyme or cleavage        substrate to the receptacle;    -   monitoring a target section of the receptacle other than the        site occupied by the label when bound to the cleavable linker;        and    -   detecting the presence of the label in the target section of the        receptacle.

In a preferred embodiment thereof, the enzyme or cleavage substratecleaves the cleavable linker, thereby releasing the label which diffusesto a section of the receptacle that is monitored for presence of thelabel. In another embodiment, the label is a fluorescent dye.

Preferred aspects of the invention include any one of the aspects of theinvention, more particularly defined by the embodiments describedherein.

In another embodiment the device is housed in an ellipse or oval shapedunit having dimensions of about 30-300 mm on the minor axis, 100-500 mmon the major axis, and a variable thickness of about 10 to 100 mm; withthe proviso that the length of the major axis is greater than the minoraxis. In a preferred embodiment, the unit has a decreasing thicknessapproaching the user, such that the screen is tilted toward the user.More particularly the unit is housed in a material with approximately2-3 mm thickness. More particularly, the housing material is plastic.

In a particular embodiment, the device described has thedesign/structural features depicted in U.S. Design Application No.29/251,820, the contents of which are incorporated by reference as ifset forth fully herein.

AIDS Diagnostic Test Device

Recent advances in CD4 diagnostic assays allow 2 simple reagents for usein identifying and counting two cell populations in whole blood, whichis compatible with a simple fluorometer with 2 channels. The use ofsimple separations mechanism, such as a hand-crank centrifuge, simplecell filters or magnetic beads, separates the cells to be counted fromthe reagents. These 3 components comprise the AIDS Diagnostic Platformfor Use in Remote Areas (ADPURA).

The key biochemical reagents for this application are the anti-CD4antibody and the anti-CD45 antibody. These two antibodies are the basisfor the PLG method (Glencross et al (2002) CD45 assisted panleucogatingfor accurate, cost effective dual platform CD4+ T cell enumeration,Cytometry Clinical Cytometry, Special Issue: CD4: 20 years and counting:50 (2) 69-77) measures CD4+ cells using CD45-expressing cells (all whiteblood cells) for normalization. Using the two antibodies together(anti-CD4 antibody and anti-CD45 antibody) makes the CD4 count as simpleas taking the ratio between CD4-expressing cells and CD45 expressingcells.

In one embodiment of the present invention, the following components areused together:

-   -   a) A fluorometer having 2 detection channels, each comprised of        an LED, an emission filter and a photodiode detector. The        fluorometer has a user-interface and data analysis on a CPU        (central processing unit) on a printed circuit board. The body        is robust and can withstand environments with wind, sand and        water present, different LEDs and emission filters, a        streamlined user interface and fewer buttons. In addition, the        fluorometer may work off of either a battery, handcrank or other        power source not requiring a plug into an electric grid        infrastructure. The fluorometer is designed for one purpose        only, in which case the user-interface is simplified to express        the result of the reading and one button only would be needed to        activate the instrument to perform a reading. Alternatively,        multiple diagnostic applications can be performed, with new        applications capable of being loaded onto the instrument using a        “thumb drive” with a USB connection.    -   b) A reagent kit, which includes CD4 antibodies, labeled with a        fluorescent dye, such as AlexaFluor 488 dye and CD45 antibodies,        labeled with a fluorescent dye spectrally distinct from the CD4        label, such as Alexa Fluor 647 dye. The antibodies would ideally        be stabilized for transport in hot or cold conditions, perhaps        lyophilized, or perhaps an azide. Some alternate        antibody-stabilization technology exists, which could be        employed. The antibodies would be at the appropriate        concentrations and in the appropriate containers to allow simple        addition to the sample by person with little scientific        training.    -   c) One of three possible cell-separation platforms:        -   a. CD45 antibody-labeled Dynal beads and DetachaBead            technology to isolate the white blood cells from the whole            cell population and then from the remaining beads using a            simple magnet.        -   b. A hand-crank centrifuge for 500 uL tubes that could be            used to pellet the cells, combined with a simple plastic            pipette to remove the supernatant and add washing solution.            The centrifuge would be similar in principle to current            hand-crank centrifuges on the market, but modified to use            500-uL plastic tubes, to be enclosed for operator safety as            well as sample integrity, to be of a size similar to current            tabletop “picofuges,” and to be compatible with field            conditions.        -   c. A filter to separate the cells from the labeled CD4 and            CD45 antibodies.    -   d) Optional accessories to this platform would include        inexpensive plastic pipettes and a water purification system,        such as those found at stores that sell camping supplies.    -   e) The invention further includes a kit comprising the above        components, with the cell-labeling method, the cell-separations        method and the fluorometer working seamlessly together.

The workflow for this application is as follows.

-   -   a. First, the blood sample is taken from the patient and        prepared for antibody staining using an accepted method.    -   b. Second, the sample is mixed with the antibodies in the        reagent kit. Fluorophore-labeled CD4 antibodies will bind to        CD4+ cells and fluorophore-labeled CD45 antibodies will bind to        CD45+ cells. If antibody-labeled beads are used for the        separation method, these will bind to the cells as well during        this step.    -   c. Third, a separation method will be employed to remove the        free labeled antibodies from the labeled cells.        -   i. If this method uses the magnetic beads, a magnet will be            used to remove the CD4+ and CD45+ cells from the sample. The            supernatant will be discarded and the beads resuspended in a            buffer. The beads will then be removed, for example, by            using DetachaBead technology, applying the magnet to pull            out the beads, leaving the labeled cells in the supernatant.        -   ii. If the method uses a centrifuge, the sample will be spun            in the centrifuge to pellet the labeled cells. The            supernatant containing free labeled antibodies will be            discarded and the cells resuspended in buffer.        -   iii. If the method uses a filter, the sample will be applied            to the filter, which will trap the labeled cells. The            flow-through will be discarded and the labeled cells            resuspended in buffer.    -   d. Fourth, the sample will be read using the fluorometer. The        labeled cells from step c will be transferred to a 500 uL clear        PCR tube and read in the fluorometer. The fluorometer will be        automated to take a reading in both channels, perform a        calculation and express the value on an LCD or similar screen as        a CD4+ count using standard nomenclature for this test.

In another embodiment the calibrators may be analytes at a knownconcentration. For example a blank containing zero analyte representingthe low end of the assay and a tube containing a high concentration ofanalyte representing the high end of the assay when incubated with theappropriate analyte detecting dye.

In another embodiment the calibrators may be a liquid or solid standardthat produces a fluorescent signal that is equal to correspondinganalyte/dye mixtures.

In another embodiment the label may be fluorescent or light scatteringnanocrystals [Yguerabide, J. and Yguerabide, E E, 2001 J. Cell BiochemSupp1.37: 71-81; U.S. Pat. Nos. 6,214,560; 6,586,193 and 6,714,299].These fluorescent nanocrystals can be semiconductor nanocrystals ordoped metal oxide nanocrystals. Nanocrystals typically are comprised ofa core comprised of at least one of a Group II-VI semiconductor material(of which ZnS, and CdSe are illustrative examples), or a Group III-Vsemiconductor material (of which GaAs is an illustrative example), aGroup IV semiconductor material, or a combination thereof. The core canbe passivated with a semiconductor overlayering (“shell”) uniformlydeposited thereon. For example, a Group II-VI semiconductor core may bepassivated with a Group II-VI semiconductor shell (e.g., a ZnS or CdSecore may be passivated with a shell comprised of YZ wherein Y is Cd orZn, and Z is S, or Se). Nanocrystals can be soluble in an aqueous-basedenvironment. An attractive feature of semiconductor nanocrystals is thatthe spectral range of emission can be changed by varying the size of thesemiconductor core.

Embodiments using in situ cleavage:

In another embodiment the instrument may measure the cleavage of areporter from a zone outside of the optical path of the instrument toone within the optical path through diffusion or active mixing. FIG. 4:Depicts a tube (306) inside the instrument. Reporter elements (302)bound to a cleavable substrate (301) are bound to the bottom of thetube. Once the substrate is cleaved then the reporter element is free(303) to travel inside the tube to within the optical path (300), of theinstrument (304) where it is detected (305).

The Reporter element may be fluorescent or colorimetric.

The Cleavable substrate may be a nucleic acid, peptide, or other organicchemical.

The cleavable substrate may be cleaved by an enzyme or a chemicalreaction.

The cleavable substrate may be attached to the tube or some otherphysical immobilizer such as magnetic or non-magnetic beads.

It is also possible that the rather than a cleavable substrate thereporter element may be displaced from a bound binder. Examples of suchare fluorescent labeled desthiobiotin which could be displaced fromStreptavidin by the higher affinity binding biotin.

Embodiments using multiple parameter device:

-   -   a. In another embodiment the machine executable instructions may        be developed by the end-user “off-line” on a computer. Such that        the name of the assay, appropriate ASEs, values and number of        relevant calibrators, curve fitting algorithm, and format of        displayed output are determined and set by the end-user and then        “uploaded” to the instrument via a connecting cable.    -   b. Once “uploaded” to the instrument the new program would        become a permanent selectable option on the instrument.

Dyes for cellular assays:

Dyes for Total Cell counting: SYTO 9, 11-18, 20-25, and 59-64, BC,TOTO-3, TO-PRO-3, DRAQ-5, Dil, DiO, WGA-546, FM1-43, Calcein AM

Dyes for Dead Cell counting: SYTOX Green, SYTOX Red, SYBR Gold, SYBRGreen, PicoGreen

Preparation of a Liquid Standard:

-   -   a. Mix sample of high concentration calibration point that is        liquid standard is intended to emulate. For example in the        Quant-iT DNA High Sensitivity Assay the high standard is 100 ng        lambda DNA plus PicoGreen dye (final concentration 0.7 uM) in a        final volume of 200 uL TE.    -   b. Read relative fluorescent value on instrument (such as the        Fluorometer described herein). For example in the Quant-iT DNA        High Sensitivity Assay read using the 460 nm excitation source.    -   c. Stepwise add concentrated stable fluorescent compound to a        diluting solution. For example in the Quant-iT DNA High        Sensitivity Assay add concentrated fluorescein (10 mM in 0.1 M        Sodium Borate, pH9 buffer) to a solution of 0.1 M Sodium Borate,        pH 9 buffer until the fluorescent signal in the device equals        that obtained in step b (approximate concentration 100 nM        fluorescein).    -   d. For all experiments going forward substitute fluorescein        solution for high standard. For example in the Quant-iT DNA High        Sensitivity Assay substitute the 100 nM fluorescein calibrator        for the calibrator containing 100 ng DNA plus PicoGreen.

In one embodiment of the present invention, the device described hereinis used for water and soil testing. In a more particular embodimentthereof the device monitors for an analyte or parameter selected fromthe group consisting of fecal coliform, pH, heavy metals, nitrates,arsenic, prions, Volatile Organic Compounds (VOC), chlorine, calcium,sodium, and glucose.

In another embodiment, the device described herein is used forinfectious disease detection and monitoring. In a more particularembodiment thereof the device monitors for analyte or parameter selectedfrom the group consisting of AIDS (CD4 assay), malaria, TB, SARS, BSE,Anthrax, Flu, Colds, Plague, and Prions.

In another embodiment, the device described herein is used for detectionor identification of biomarkers. In a more particular embodiment thereofthe biomarker is indicative of: cancer, mycoplasma, pregnancy,telomerase, antibodies, and genetic diseases.

In another embodiment, the device described herein is used for detectionor identification of enzyme substrates. In a more particular embodimentthereof, the enzyme substrate is selected from the group consisting of:nucleases, phosphotases, glycoases, kinases, proteases and peroxidases.

In another embodiment, the device described herein is used for cellbiology reagent validation and quality control (QC). In a moreparticular embodiment thereof the device monitors for an analyte orparameter selected from the group consisting of sodium, calcium,glucose, magnesium, potassium, zinc, thallium, pH, oxygen, nitric oxide,carbon dioxide, chloride and enzyme substrates (nucleases, phosphotases,glycoeases, kinases, proteases and peroxidases).

In another embodiment, the device described herein is used for bacterialdetermination. In a more particular embodiment thereof the devicemonitors for analytes associated with red tide or E. Coli.

In another embodiment, the device described herein is used in the fieldof cosmetics. In a more particular embodiment thereof, the devicemonitors for Reactive Oxygen Species (ROS), bacterial contamination,live/dead cells, melamine determination, cholesterol

In another embodiment, the device monitors for GFP, chlorophyll, orbiowarfare agents.

In another embodiment, the device described herein is used forautomotive purposes. More particularly, leak detection, oil detection,air conditioning, coolant detection.

In another embodiment, the device described herein is used in forensics;more particularly sample determination, such as thedetection/quantification of blood, urine or sperm.

In another embodiment, the device described herein is used foragricultural detection/quantitation. More particularly, for thedetection/quantitation of enzyme substrates, such as, phytases,cellulases and others enzyme substrates described herein.

In another embodiment, the device described herein is used in end-pointPCR. More particularly, the device monitors/detects stains including,SYBR Green, PicoGreen, SYBR Gold, among others. In another embodiment,the device detects/monitors for molecular beacons. In another embodimentthe device detects/monitors for isothermal amplification.

The present invention relates to devices and methods for quantifyingmultiple analytes. The terms “quantify” or “measure the quantity” asused herein are interchangeable. The quantitative measurement can be anymeasurement designed to provide the end-user with information regardingthe amount of the analyte in the sample. Thus the measurement may be anabsolute measurement of the analyte, such as mass, or the measurementmay be a relative measurement, such as concentration or parts permillion, etc. Of course, the quantity of analyte may be equal to zero,indicating the absence of the analyte sought, or that the analyte isbelow the detectable level of the assay as measured by the instrument.The quantity may simply be the measured energy as detected by thephotodetector, without any additional measurements or manipulations.Alternatively, the quantity may be expressed as a difference, percentageor ratio of the measured value of the analyte to a measured value ofanother compound including, but not limited to, a standard. The quantitymay even be expressed as a difference or ratio of the analyte to itself,measured at a different point in time.

The quantity of analyte may be determined directly from the detectedenergy value, or the detected energy value may be used in an algorithm,with the algorithm designed to correlate the detected energy value tothe quantity of analyte in the sample. To that end, the machineexecutable instructions may also be configured to determine the quantityof the analyte based upon the detected energy value.

In one particular embodiment, the machine executable instructionsperform a method of calculating the quantity of an analyte in a sampleby generating a fluorescence standard curve and correlating thefluorescence intensity of the sample to a quantity of analyte using thisfluorescence standard curve. In a more particular embodiment, thefluorescence standard curve can be generated by measuring thefluorescence intensity of a “blank” sample, i.e., a sample known to bewithout the analyte, (g) and measuring the fluorescence intensity ofonly one standard containing a known amount of the analyte (v). Ofcourse, the fluorescence standard curve could be generated using morethan one standard containing a known amount of the analyte. Once thefluorescence value of the blank and standard have been measured, themachine executable instructions can then generate the fluorescencestandard curve. The fluorescence standard curve may have a predetermineddegree of sigmoidicity (n) and curvature (k), prior to the measuring theblank and standard(s), and the machine executable instructions maypossess these (k) and (n) values as part of the algorithm for generatingthe fluorescence standard curve. Once the machine executableinstructions generate the fluorescence standard curve, the fluorescenceintensity of a sample (y) can be measured and the (y) value can becorrelated to an analyte quantity using the fluorescence standard curve.

In a specific embodiment, the fluorescence standard curve can becharacterized by the following equation:

y=r(x ^(n)/(x ^(n) +k))+g;  (I)

-   -   where (r) is a correctional value determined by the formula:

r=(v−g)((s ^(n) +k)/s ^(n))  (II)

and where (s) is the quantity of analyte in the high-end standard.

In an even more specific embodiment, the value of (n) in equations I andII approach or are approximately equal to 1 (one). Values of n includebut are not limited to, 0≦n≦10, 0≦n≦5, 0.5≦n≦3, 0.75≦n≦1.5, 0.8≦n≦1.2,0.9≦n≦1.1 and 0.95≦n≦1.05. In addition, the curve may approachlinearity. As k approaches infinity, the curve approaches linearity. Itis readily understood what is meant when a curve “approaches linearity.”

In another specific embodiment, three standards are used, where onestandard is a blank, another standard is a mid-range standard and thethird standard is the high-end standard. In this particular embodiment,(n) is predetermined, but (k) may be variable and thus should bedetermined. In this embodiment, k is solved for using equation IV below.

k=[s ^(n) t ^(n)(d−1)]/[s ^(n)−(t ^(n) d)]  (IV)

The value d is a correctional value that is equal to the ratio of thefluorescence value of the two non-blank background-corrected non-blankstandards that is solved for using equation V below.

d=(v−g)/(w−g)  (V)

In equation V, (w) is the fluorescence intensity of the mid-rangestandard used in this assay, value (v) is the fluorescence intensity ofthe high-end standard and (g) is the fluorescence intensity of theblank. In equation IV, the (t) is the quantity of analyte in themid-range standard.

Another embodiment provides a method of calculating the ratio of twoanalytes in a sample container, said method comprising

-   -   a) generating a fluorescence standard curve for the two analytes        comprising: measuring the fluorescence intensity of a first        blank analyte sample (g1) and a second analyte analyte sample        (g2) and measuring the fluorescence intensity of at least one        high-end standard for the first analyte (v1) and at least one        high-end standard for the second analyte (v2), wherein said        curve correlates fluorescence intensity to each analyte quantity        or relative quantity, and wherein said curves have a        predetermined degree of sigmoidicity (n) and curvature (k);    -   b) measuring the fluorescence intensity of said samples (y1 and        y2), wherein said sample comprises a fluorescent moiety capable        of indicating the presence of said analytes in said sample; and    -   c) correlating said fluorescence intensity in said samples (y1        and y2) to the quantity of said analyte using said fluorescence        standard curve.

Thus the present invention relates to a device for quantifying ananalyte, with the device comprising machine executable instructions thatimplement the methods of quantifying an analyte utilizing a fluorescencestandard curve equation I, above. This device may, of course, furthercomprise each of the components listed herein, such as, but not limitedto, a receptacle for holding a sample container, photodetector, and oneor more ASEs.

The computer processing unit may also comprise sufficient memory andinstructions or operations for associating a sample identity tag with aparticular sample. In this embodiment, the device may comprise a meansfor identifying an identity tag associated with a sample container. Inone particular embodiment, the sample identity tag is machine readable.Examples of identity tags include, but are not limited to, barcodes,data matrix barcodes, radio frequency identity tags, optical tags andthe like. The means for identifying the identity tag should, of course,be suited to the type of identity tag employed. In turn, the machineexecutable instructions may then be able to match the determinedquantity value of the analyte with a particular sample, based on thesample identity tag.

The devices of the present invention may also comprise a user interface.Various user interfaces can be provided to facilitate user control andto enhance operability of the devices. Input interfaces include, but arenot limited to, data entry devices such as a keyboard, keypad,touch-screen display, mouse, voice recognition input, or other dataentry device. In one specific embodiment, the user interface comprises anon-numerical keypad. Output interfaces include, but are not limited to,a display screen, monitor, a printer, a speaker or other output device.In another specific embodiment, the user interface comprises a displayscreen. In yet another specific embodiment, the user interface comprisesboth a non-numerical keypad and a display screen. In a more specificembodiment, the user interface is configured to allow the end-user toselect the analyte being quantified. Once the analyte is selected by theend-user, the machine executable instructions can then determine whichASE to employ, if necessary, to quantify the analyte.

The devices of the present invention may optionally comprise an internalpower source, used to power the various components of the device. In oneembodiment, the power source is rechargeable. In another embodiment, theinternal power source is not rechargeable.

In another embodiment, the devices of the present invention may compriseone or more communications ports. The communications port(s) is (are)capable of connecting to another device such as, but not limited to, acomputer, a disk drive, a flash memory drive, a monitor, a printer,another similar device for quantifying analytes. For example, thefunctions of the device can be updated or altered, and the device can becalibrated or recalibrated with new machine executable instructions froma computer, CD or DVD via the communications port. The devices are notlimited by the types of communications ports. Examples of communicationsports include bur are not limited to universal serial bus (USB), anaudio/video serial bus (IEEE 1394) (“firewire”), and infrared port and aradio frequency port. Radio ports include Bluetooth® ports, Wi-Fi portsand the like.

In a particular embodiment of any of the aforementioned embodiments, theanalyte is a eukaryotic or prokaryotic cell.

In another embodiment, the device or method described herein is used forquantification of DNA at low levels in liquid samples. In anotherembodiment the device or method described herein is used forquantification of DNA at broad ranges in a liquid sample. In anotherembodiment, the device or method is used for quantification of RNA inliquid samples. In another embodiment, the device or method is used forquantification of protein in liquid samples. In another embodiment, thedevice or method is used for quantification of live:dead cells in liquidsamples. In another embodiment, the device or method is used forquantification of GFP expression levels in liquid samples. In anotherembodiment, the device or method is used for detection andquantification of Mycoplasma contamination of cell cultures and cellculture reagents. Further description of these embodiments are describedin the Examples that follow.

EXAMPLES Example 1 Analyzing RNA Concentrations

Using Equation I: y=r(x^(n)/(x^(n)+k))+g;

-   -   where (r) is a correctional value determined by the formula:

r=(v−g)((s ^(n) +k)/s ^(n))  (II)

the concentration of RNA in a sample is determined. The value (y) is thefluorescence intensity of the sample with an unknown concentration ofanalyte. The high-end standard, which is the value of (s), has aconcentration of 500 ng/ml. The sigmoidicity (n) is set to a value of1.10 and the curvature (k) is set to 2350. The fluorescence intensity ofthe blank (g) is 22.16 relative fluorescence units (RFU) and thefluorescence intensity of the high-end standard (v) is 543.97 RFU. Thesevalues are used in equation II to solve for r, which is 1839.20

Solving equation I for x, which is, in this example, the concentrationof RNA in the sample, results in Equation III

x=|(y−g))/(r−(y−g)|^(1/n)  (III)

Using a fluorometer of the present invention, the fluorescence intensityof a sample containing 400 ng/ml of RNA is measured as 459.40 RFU.Plugging this value into equation III, the fluorometer obtains aconcentration value of RNA in the sample of 402.34 ng/ml.

Example 2 Analyzing DNA Concentrations at Low Levels

Using equations I, II and II from above, the concentration of low levelsof DNA can also be assessed. In this example, the (n) is set to 1.00 and(k) is set to 9999999. The blank has a fluorescence value (g) of 7.61RFU and the high-end standard has a fluorescence value (v) of 3020.30RFU. The high-end standard (s) has a concentration of 500 ng/ml. Usingthese values in Equation II, the correctional value (r) is determined tobe 60256806.66.

The sample contains 400 ng/ml of DNA and has a fluorescence value (y) of2401.60. The concentration of DNA, using Equation III is determined tobe 397.31 ng/ml.

Example 3 Analyzing DNA over a Broad Range of Concentrations

Using equations I, II and II from above, the concentration of DNA canalso be assessed. In this example, the (n) is set to 1.00 and (k) is setto 22.5. The blank has a fluorescence value (g) of 25.36 RFU and thehigh-end standard has a fluorescence value (v) of 2213.20 RFU. Thehigh-end standard (s) has a concentration of 5 μg/ml. Using these valuesin Equation II, the correctional value (r) is determined to be 12033.12.

The sample contains 4.0 μg/ml of DNA and has a fluorescence value (y) of1841.10. The concentration of DNA, using Equation III is determined tobe 4.0 μg/ml.

Example 4 Analyzing Protein Concentrations

Using equations I, II and III from above, the concentration of proteincan also be assessed. When assessing protein concentration, it may bedesirable to use three standards rather than two standards as above.

If three standards are used, then k may be variable, and if k isvariable, the algorithm will first solve for k using the threestandards. Once k is solved for, the algorithm uses this value inequations I, II and III above. To solve for k when k is variable, usingthree standards, the follow equation is employed:

k=[s ^(n) t ^(n)(d−1)]/[s ^(n)−(t ^(n) *d)]  (IV)

where d is a correctional value that is equal to the ratio of thefluorescence value of the two non-blank background-corrected standardsthat is determined by equation V.

d=(v−g)/(w−g)  (V)

In equation V, w is the fluorescence value of the mid-range standardused in this assay. Once the correctional value d is determined, thisvalue is plugged into equation IV to solve for (k). The value (s) is theconcentration of the high-end standard, as in equation II, and (t) isthe concentration of the mid-range standard, and (n) is predetermined.Once (k) is solved for, using equations IV and V, the fluorescence valueof the unknown is measured (y) and the concentration of the unknown (x)is determined using equation III, as above.

In this example, the (n) is set to 2.15. The blank has a fluorescencevalue (g) of 42 RFU and the high-end standard has a fluorescence value(v) of 3230 RFU. The high-end standard (s) has a concentration of 5.000μg/200 μl. The mid-range standard has a fluorescence value (w) of 1286and a concentration (t) of 2.000 μg/200 μl. Using these values inEquations IV and V, (k) equals 10.79. Using k=10.79 and n=2.15 inequation II, the correctional value (r) (based on the high-end standard)equals 4371.28. Finally, the fluorescence value of a sample (y)containing 3.000 g/200 μl of protein is measured as 2334. Thefluorometer, employing the algorithms described herein determines thatthe concentration of the sample is 3.2 μg/200 μl.

In the following Examples “Quant-it” assay firmware selections areconnected to analyte sensing elements with predetermined algorithms,standards, light sources, filters, etc., as described above for thevarious types of analytes. These coordinated analyte sensing elementsmake the device very easy for the technician to operate.

Example 5 Quantification of DNA at Low Levels in Liquid Samples

PicoGreen dye, is diluted in a specific buffer to make a Working DyeSolution of 0.7 μM. Between 180 and 199 uL of the Working Dye Solutionis added to a 500 uL clear plastic PCR tube, described herein. Two ofthe tubes are used for standards to calibrate the instrument for theassay. 10 uL of a solution of TE (10 mM Tris, 1 mM EDTA) is added as the“zero” to one tube. 10 uL of a 10 ng/uL solution of lambda DNA in TE isadded to the second tube and is the high standard. To the remainingtubes, between 1 and 20 uL of an unknown sample is added, for a finalconcentration of 200 uL in each tube. Each of the tubes is mixed using avortexer or by inverting the tube and then incubated at room temperaturefor 2 minutes. To determine the amount of DNA in the assay tube, theuser chooses the “Quant-iT DNA HS” assay using the firmware built intothe fluorometer, and then follows prompts to allow the fluorometer toread Standard 1 (the zero standard described above), Standard 2 (thehigh standard described above) and then any number of samples. Using thetwo standards, the instrument determines the concentration of DNA in thesample tubes using the equations described in Example 2. Theconcentration of DNA in the solution in the assay tube is displayed onthe screen of the fluorometer. An option is included in the firmware toperform a dilution calculation by asking the user to choose from 1, 2,3, 4, 5, 10, 15 or 20 uL of the sample added to the sample tube. Theconcentration of DNA in the original sample tube is then displayed onthe screen of the fluorometer. All of these data can be simultaneouslytransferred to a computer using the compatible ports and data loggingsoftware, described herein.

Example 6 Quantification of DNA at Broad Ranges in Liquid Samples

HiQuant dye, is diluted in a specific buffer to make a Working DyeSolution of 2 μM. Between 180 and 199 uL of this Working Dye Solution isadded to a 500 uL clear plastic PCR tube, described herein. Two of thetubes are used for standards to calibrate the instrument for the assay.10 uL of a solution of TE (10 mM Tris, 1 mM EDTA) is added as the “zero”to one tube. 10 uL of a 100 ng/uL solution of lambda DNA in TE is addedto the second tube to serve as the high standard. To the remainingtubes, between 1 and 20 uL of an unknown sample is added, for a finalconcentration of 200 uL in each tube. Each of the tubes is mixed using avortexer or by inverting the tube and then incubated at room temperaturefor 2 minutes. To determine the amount of DNA in the assay tube, theuser chooses the “Quant-iT DNA BR” assay using the firmware built intothe fluorometer, and then follows prompts to allow the fluorometer toread Standard 1 (the zero standard described above), Standard 2 (thehigh standard described above) and then any number of samples. Using thetwo standards, the instrument determines the concentration of DNA in thesample tubes using the equations described in Example 3. Theconcentration of DNA in the solution in the assay tube is displayed onthe screen of the fluorometer. An option is included in the firmware toperform a dilution calculation by asking the user to choose from 1, 2,3, 4, 5, 10, 15 or 20 uL of the sample added to the sample tube. Theconcentration of DNA in the original sample tube is then displayed onthe screen of the fluorometer. All of these data can be simultaneouslytransferred to a computer using the compatible ports and data loggingsoftware, described herein.

Example 7 Quantification of RNA in Liquid Samples

RiboRed dye, is diluted in a specific buffer to make a Working DyeSolution of 0.04 μM. Between 180 and 199 uL of this Working Dye Solutionis added to a 500 uL clear plastic PCR tube, described herein. Two ofthe tubes are used for standards to calibrate the instrument for theassay. 10 uL of a solution of TE (10 mM Tris, 1 mM EDTA) is added as the“zero” to one tube. 10 uL of a 10 ng/uL solution of ribosomal RNA in TEis added to the second tube and will be the high standard. To theremaining tubes, between 1 and 20 uL of an unknown sample is added, fora final concentration of 200 uL in each tube. Each of the tubes is mixedusing a vortexer or by inverting the tube and then incubated at roomtemperature for 2 minutes. To determine the amount of RNA in the assaytube, the user chooses the “Quant-iT RNA” assay using the firmware builtinto the fluorometer, and then follows prompts to allow the fluorometerto read Standard 1 (the zero standard described above), Standard 2 (thehigh standard described above) and then any number of samples. Using thetwo standards, the instrument determines the concentration of RNA in thesample tubes using the equations described in Example 2. Theconcentration of RNA in the solution in the assay tube is displayed onthe screen of the fluorometer. An option is included in the firmware toperform a dilution calculation by asking the user to choose from 1, 2,3, 4, 5, 10, 15 or 20 uL of the sample added to the sample tube. Theconcentration of RNA in the original sample tube is then displayed onthe screen of the fluorometer. All of these data can be simultaneouslytransferred to a computer using the compatible ports and data loggingsoftware, described herein.

Example 8 Quantification of Protein in Liquid Samples

NanoOrange dye, is diluted in a specific buffer to make a Working DyeSolution of 4 μM. Between 180 and 199 uL of this Working Dye Solution isadded to a 500 uL clear plastic PCR tube, described herein. Three of thetubes are used for standards to calibrate the instrument for the assay.10 uL of a solution of TE (10 mM Tris, 1 mM EDTA) is added as the “zero”to one tube. 10 uL of a 200 ng/uL solution BSA in TE is added to thesecond tube and will be the middle standard. 10 uL of a 400 ng/uLsolution BSA in TE is added to the third tube and will be the highstandard. To the remaining tubes, between 1 and 20 uL of an unknownsample is added, for a final concentration of 200 uL in each tube. Eachof the tubes is mixed using a vortexer or by inverting the tube and thenincubated at room temperature for 15 minutes. To determine the amount ofprotein in the assay tube, the user chooses the “Quant-iT Protein” assayusing the firmware built into the fluorometer, and then follows promptsto allow the fluorometer to read Standard 1 (the zero standard describedabove), Standard 2 (the middle standard described above), Standard 3(the high standard described above) and then any number of samples.Using the three standards, the instrument determines the concentrationof protein in the sample tubes using the equations described in Example4. The concentration of protein in the solution in the assay tube isdisplayed on the screen of the fluorometer. An option is included in thefirmware to perform a dilution calculation by asking the user to choosefrom 1, 2, 3, 4, 5, 10, 15 or 20 uL of the sample added to the sampletube. The concentration of protein in the original sample tube is thendisplayed on the screen of the fluorometer. All of these data can besimultaneously transferred to a computer using the compatible ports anddata logging software, described herein.

Example 9 Quantification of Cells (Eukaryotic or Prokaryotic) in LiquidSamples

SYTO 9 dye, is diluted in a specific buffer to make a Working DyeSolution of 0.02 mM. Between 180 and 199 uL of this Working Dye Solutionis added to a 500 uL clear plastic PCR tube, described herein. Two ofthe tubes are used for standards to calibrate the instrument for theassay. 200 uL of a solution of water is added as the “zero” to one tube.200 uL of a 100 nM fluorescein solution in 0.1 M Sodium Borate, pH 9buffer is added to the second tube and will be the high standard. To theremaining tubes, between 1 and 20 uL of an unknown sample is added, fora final concentration of 200 uL in each tube. Each of the tubes is mixedusing a vortexer or by inverting the tube and then incubated at roomtemperature for 5 minutes. To determine the amount of cells in the assaytube, the user chooses the “Quant-iT Cell Count” assay using thefirmware built into the fluorometer, and then follows prompts to allowthe fluorometer to read Standard 1 (the zero standard described above),Standard 2 (the high standard described above) and then any number ofsamples. Using the two standards, the instrument determines theconcentration of protein in the sample tubes using the equationsdescribed herein (see equations I-V). The concentration of cells in thesolution in the assay tube is displayed on the screen of thefluorometer. An option is included in the firmware to perform a dilutioncalculation by asking the user to choose from 1, 2, 3, 4, 5, 10, 15 or20 uL of the sample added to the sample tube. The concentration of cellsin the original sample tube is then displayed on the screen of thefluorometer. All of these data can be simultaneously transferred to acomputer using the compatible ports and data logging software, describedherein.

Example 10 Quantification of Live:Dead Ratios of Cells (Eukaryotic orProkaryotic) in Liquid Samples

SYTO 9 dye and SYTOX Red dye, is diluted in a specific buffer to make aWorking Dye Solution of 0.02 mM of each dye. Between 180 and 199 uL ofthis Working Dye Solution is added to a 500 uL clear plastic PCR tube,described herein. Two of the tubes are used for standards to calibratethe instrument for the assay. 200 uL of a solution of water is added asthe “zero” to one tube. 200 uL of a 100 nM fluorescein dye and 100 nMAlexa Fluor 647 dye solution in 0.1M Sodium Borate, pH 9 buffer is addedto the second tube and will be the high standard. To the remainingtubes, between 1 and 20 uL of an unknown sample is added, for a finalconcentration of 200 uL in each tube. Each of the tubes is mixed using avortexer or by inverting the tube and then incubated at room temperaturefor 5 minutes. To determine the amount of live and dead cells in theassay tube, the user chooses the “Quant-iT Live/Dead” assay using thefirmware built into the fluorometer, and then follows prompts to allowthe fluorometer to read Standard 1 (the zero standard described above),Standard 2 (the high standard described above) and then any number ofsamples using both a “blue” channel (Ex/Em: 460 nm/520 nm) and a deepred channel (Ex/Em: 630 nm/650 nm) as described in [0031]. Using the twostandards, the instrument determines the relative concentration of totalcells and dead cells in the sample tubes using the equations describedherein. The ratio of the two is displayed on the screen of thefluorometer. All of these data can be simultaneously transferred to acomputer using the compatible ports and data logging software, describedherein.

Example 11 Quantification of GFP Expression Levels in Cells in a LiquidSamples

SYTO 61 dye, is diluted in a specific buffer to make a Working DyeSolution of 0.02 mM. Between 180 and 199 uL of this Working Dye Solutionis added to a 500 uL clear plastic PCR tube, described in [0016]. Two ofthe tubes are used for standards to calibrate the instrument for theassay. 200 uL of a solution of water is added as the “zero” to one tube.200 uL of a 100 nM fluorescein dye and 100 nM Alexa Fluor 647 dyesolution in 0.1 M Sodium Borate, pH 9 buffer is added to the second tubeand will be the high standard. To the remaining tubes, between 1 and 20uL of an unknown sample is added, for a final concentration of 200 uL ineach tube. Each of the tubes is mixed using a vortexer or by invertingthe tube and then incubated at room temperature for 5 minutes. Todetermine the amount of cells in the assay tube, the user chooses the“Quant-iT GFP” assay using the firmware built into the fluorometer, andthen follows prompts to allow the fluorometer to read Standard 1 (thezero standard described above), Standard 2 (the high standard describedabove) and then any number of samples using both a “blue” channel(Ex/Em: 460 nm/520 nm) and a deep red channel (Ex/Em: 630 nm/650 nm) asdescribed in [0031]. Using the two standards and the signal from theSYTO 61, the instrument determines the concentration of cells in thesample tubes using the equations described in [0066]. Using the twostandards and the signal from the “blue” channel, the instrumentdetermines the concentration of GFP in the sample tube using theequations described in [0066]. The ratio of GFP to cell concentration inthe solution in the assay tube is displayed on the screen of thefluorometer. All of these data can be simultaneously transferred to acomputer using the compatible ports and data logging software, describedherein.

Example 12 Detection and Quantification of Mycoplasma Contamination ofCell Cultures, and Cell Culture Reagents

GFP is covalently attached to one end of a peptide that is bound to thebottom of a 500 uL clear plastic tube. Between 180 and 199 uL of thisWorking Solution is added to a 500 uL clear plastic PCR tube, describedherein. Two of the tubes are used for standards to calibrate theinstrument for the assay. 200 uL of a solution of water is added as the“zero” to one tube. 200 uL of a 100 nM fluorescein solution in 0.1MSodium Borate, pH 9 buffer is added to the second tube and will be thehigh standard. To the remaining tubes, between 1 and 20 uL of an unknownsample is added, for a final concentration of 200 uL in each tube. Eachof the tubes is mixed using a vortexer or by inverting the tube and thenincubated at room temperature for 5 minutes. To determine the presenceof Mycoplasma contamination, the user chooses the “Quant-iT Mycoplasma”assay using the firmware built into the fluorometer, and then followsprompts to allow the fluorometer to read Standard 1 (the zero standarddescribed above), Standard 2 (the high standard described above) andthen any number of samples. Using the two standards, the instrumentdetermines the concentration of Mycoplasma in the sample tubes using theequations described herein. The concentration of Mycoplasma in thesolution in the assay tube is displayed on the screen of thefluorometer. An option is included in the firmware to perform a dilutioncalculation by asking the user to choose from 1, 2, 3, 4, 5, 10, 15 or20 uL of the sample added to the sample tube. The concentration ofMycoplasma in the original sample tube is then displayed on the screenof the fluorometer. All of these data can be simultaneously transferredto a computer using the compatible ports and data logging software,described herein.

Example 13 Prokaryotic Live:Dead Determination

2 mL of S. aureus are collected into microcentrifuge tube and pellet.One tube is treated with 70% IPA (Isopropyl Alcohol) ˜30 min RT to killcells and then Washed/Spinned 2×. Pellets are resuspended andtransferred into 10 mL 0.85% NaCl. Test solutions are prepared withratios depicted in Table 1, and appropriate stain is added: 0.3 uL of3.35 mM SYTO 9 and/or 1 uL of 1 mM SYTOX Red. The solutions areincubated for 15 min at RT, 200 uL are transferred to a PCR tube andanalyzed.

TABLE 1 Live:Dead Ratio 1 2 3 4 A 100:0  100:0  100:0  100:0  B 50:5050:50 50:50 50:50 C  0:100  0:100  0:100  0:100 Stain→ No Stain SYTO 9SYTOX SYTO 9 & Red SYTOX Red

Results:

Blue Excitation—SYTO 9 shows little change in signal as the ratio ofLive:Dead cells decreases. SYTOX Red is not optimally excited by 460 nmexcitation (see FIG. 5A).

Red Excitation—SYTOX Red signal increases as the ratio of Live:Deadcells decrease. SYTO 9 is not optimally excited by 460 nm excitation(see FIG. 5B).

Ratio Determination—As the ratio of Live:Dead cells decreases the SYTO 9signal decreases slightly, while the SYTOX Red signal increasessignificantly as it stains the dead cells. The ratio between the signalfrom the 460 nm source (exciting only SYTO 9) and the signal from the630 nm source (exciting only SYTOX Red) decreases proportionally to thelive:dead ratio of the cells (see FIG. 5C).

Example 14 Eukaryotic Cell Counting

Eukaryotic Cell Line (Jurkat, MRCS, U2OS, 3T3, BPAE, COS, HeLa, CHO-K₁)are collected and cell concentration are counted and set equal (1×10⁶cells/mL) using the Coulter Counter. 1 mL of cells are mixed with SYTO 9(final concentration 1 uM) for 15 minutes at RT. 200 uL of the solutionis transferred to a PCR tube and read using 460 nm excitation.

Results: 460 nm excitation—SYTO 9 shows a fluorescent response for alleukaryotic cell lines tested significantly above background (see FIG.6).

Each of the aforementioned references are hereby incorporated byreference as is set forth fully herein.

1. A device for measuring the quantity of one or more predeterminedanalytes, wherein the device is an integrated unit which comprises areceptacle for holding a sample container having an analyte, aphotodetector, one or more distinct and operably linked analyte sensingelements (ASE) and a computer processing unit with machine executableinstructions, wherein the ASE comprises: a) an energy source forexciting the sample, wherein the energy source is configured to emit apredetermined peak wavelength of light; b) an excitation filter, whereinthe excitation filter is configured to isolate a predetermined range ofwavelengths of light from the energy source; c) an emission filter,wherein the emission filter is configured to isolate a predeterminedrange of wavelengths of light emitted from the excited sample; andwherein each of the ASE is configured to measure the quantity of thepredetermined analyte and wherein the machine executable instructionsare configured to select the proper ASE corresponding to the analyte tobe measured.
 2. The device of claim 1, wherein the energy source is alight emitting diode (LED).
 3. The device of claim 1, wherein thepredetermined analyte is selected from the group consisting of DNA, RNA,protein, eukaryotic or prokaryotic cells, carbohydrates, lipids,viruses, pH and metals ions.
 4. The device of claim 1, wherein themachine executable instructions are further configured to determine theconcentration of the specific analyte based upon an emitted light fromthe excited sample.
 5. The device of claim 1, wherein the device furthercomprises a user interface.
 6. The device of claim 5, wherein the userinterface comprises a display and a non-numerical keypad.
 7. The deviceof claim 1, wherein the sample container receptacle is configured to fitan optically clear 0.5 microcentrifuge tube.
 8. The device of claim 1,wherein the device further comprises an internal power source.
 9. Thedevice of claim 1, further comprising at least one communications port.10. The device of claim 9, wherein the communications port is selectedfrom the group consisting of a universal serial bus (USB) port, anaudio/video serial bus (IEEE 1394), an infrared (IR) port and a radiofrequency (RF) port.
 11. The device of claim 1, wherein the devicecomprises a first and second ASE.
 12. The device of claim 11, whereinthe first ASE comprises a LED that emits light with a peak wavelength ofabout 470 nm, an excitation filter that filters out light with awavelength of greater than about 490 nm and an emission filter thatfilters out light with a wavelength of less than about 520 nm andgreater than about 580 nm.
 13. The device of claim 11, wherein thesecond ASE comprises a LED that emits light with a peak wavelength ofabout 640 nm, an excitation filter that filters out light with awavelength of less than about 570 and greater than about 647 nm and anemission filter that filters out light with a wavelength of less thanabout 652 nm.
 14. The device of claim 5, wherein the user interface isconfigured to allow a user to select the analyte for measurement. 15.The device of claim 1, wherein the machine executable instructions arecapable of selecting the analyte for measurement without user input. 16.The device of claim 1, wherein dimensions of the device are about 30-300mm on a minor axis, 100-500 mm on a major axis, and a variable thicknessof about 10 to 100 mm; with the proviso that the dimension of the majoraxis is greater than the minor axis.
 17. A method of calculating thequantity of an analyte in an optically clear sample container, themethod comprising a) generating a fluorescence standard curve comprisingmeasuring the fluorescence intensity of a blank sample (g) and measuringthe fluorescence intensity of at least one high-end standard (v),wherein the curve correlates fluorescence intensity to analyte quantity,and wherein the curve has a predetermined degree of sigmoidicity (n) andcurvature (k); b) measuring the fluorescence intensity of the sample(y), wherein the sample comprises a fluorescent moiety capable ofindicating the presence of the analyte in the sample; and c) correlatingthe fluorescence intensity in the sample (y) to the quantity of theanalyte using the fluorescence standard curve.
 18. The method of claim17, wherein the curve is characterized by the equation:y=r(x ^(n)/(x ^(n) +k))+g;  (I) wherein r is a correctional valuedetermined by the formula:r=(v−g)((s ^(n) +k)/s ^(n))  (II) wherein (s) is the quantity of analytein the high-end standard.
 19. A method for detecting the presence of apredetermined analyte in a sample wherein the sample is in an opticallyclear sample container, the method comprising: a) contacting the samplewith a reporter molecule to form a contacted sample: b) detecting thepresence of the predetermine analyte, wherein detecting comprisesplacing the optically clear container in a receptacle for holding thesample container of an integrated device for the measurement of apredetermined analyte, wherein the device further comprises: i) aphotodetector, one or more distinct and operably linked analyte sensingelements (ASE) and a computer processing unit with machine executableinstructions, wherein the ASE comprises: an energy source for excitingthe sample, wherein the energy source is configured to emit apredetermined peak wavelength of light; an excitation filter, whereinthe excitation filter is configured to isolate a predetermined range ofwavelengths of light from the energy source; an emission filter, whereinthe emission filter is configured to isolate a predetermined range ofwavelengths of light emitted from the excited sample; and wherein eachof the ASE is configured to measure the quantity of the predeterminedanalyte and wherein the machine executable instructions are configuredto select the proper ASE corresponding to the analyte to be measured.20. A method of calculating the ratio of two analytes in a samplecontainer, the method comprising a) generating a fluorescence standardcurve for the two analytes comprising: measuring the fluorescenceintensity of a first blank analyte sample (g1) and a second analytesample (g2) and measuring the fluorescence intensity of at least onehigh-end standard for the first analyte (v1) and at least one high-endstandard for the second analyte (v2), wherein the curve correlatesfluorescence intensity to each analyte quantity or relative quantity,and wherein the curves have a predetermined degree of sigmoidicity (n)and curvature (k); b) measuring the fluorescence intensity of thesamples (y1 and y2), wherein the sample comprises a fluorescent moietycapable of indicating the presence of the analytes in the sample; and c)correlating the fluorescence intensity in the samples (y1 and y2) to thequantity of the analyte using the fluorescence standard curve.